Therapeutic compositions and methods for antibody and Fc-containing targeting molecule-based targeted delivery of bioactive molecules by bacterial minicells

ABSTRACT

The present application relates to the use of bacterial minicells as targeted delivery agents in vivo and in vitro. Described herein are genetically engineered eubacterial minicells designed to preferentially target and deliver therapeutically relevant agents using a minicell surface coupling molecule capable of binding and displaying antibodies or other Fc-containing targeting moiety fusions and conjugates.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Nos. 61/442,999, filed Feb. 15, 2011, and61/526,219, filed Aug. 22, 2011. The content of these relatedapplications are herein expressly incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSEQLISTING.TXT, created Feb. 14, 2012, which is 248 Kb in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

BACKGROUND

Field of the Invention

The present application is drawn to compositions and methods for theproduction, purification, formulation, and use of eubacterial minicellsas targeted delivery vehicles for in vivo and in vitro nucleic acid,protein, radionuclide, and small molecule drug delivery for theinhibition or prevention of disease as well as a targeted in vivoimaging and diagnostic technology.

Description of the Related Art

The following description of the background of the invention is providedto aid in understanding the invention, but is not admitted to describeor constitute prior art to the invention. The contents of the articles,patents, and patent applications, and all other documents andelectronically available information mentioned or cited in thisapplication, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other documents.

The need for a robust delivery vehicle capable of encapsulation of awide array of bioactive molecule species that is also capable ofselectively targeting specific cell, organ, and tissue types issignificant. Many molecular therapies are hampered by one or more invivo limitations that include (i) adverse toxic side effects due toon-target or off-target effects on healthy cells, organs, and tissues,(ii) poor pharmacokinetics (PK) and (iii) poor uptake into cells. Thetargeted delivery of cytotoxic drugs, imaging agents, therapeuticnucleic acids, and other biologically active therapeutic moleculesdirectly into the site(s) and cells that cause disease could relievemany of these deficiencies by decreasing on-target or off-target toxiceffects exerted on non-disease tissue, improving the pharmacokinetics oftherapeutic agents allowing for more effective administration, andenhancing uptake into cells. Accordingly, it is well recognized that thedevelopment of therapies targeted to specific cell, organ, and tissuetypes represents an important new frontier for clinically relevanttherapeutic, diagnostic, theranostic, and imaging technologies.

In the case of chemotherapeutic agents (e.g., small molecule cytotoxicdrugs) and protein toxins used in the treatment of most cancers,efficacy of the chemotherapeutic agent or protein toxin is significantlylimited by toxicity to normal tissues. In addition, drug pharmacokinetic(PK) parameters contributing to systemic exposure levels frequently arenot and cannot be fully optimized to simultaneously maximize anti-tumoractivity and minimize side-effects, particularly when the same cellulartargets or mechanisms are responsible for anti-tumor activity and normaltissue toxicity. This results in a very narrow therapeutic index, commonfor most cytotoxic chemotherapeutics and protein toxins.

One way to enhance the therapeutic index of existing drugs is to bind,conjugate, or package them so that a larger percentage of theadministered dose ends up in the vicinity of the tumor (passivetargeting) and/or inside the tumor cells (active targeting). Manydifferent approaches to targeted delivery have been taken to datealthough few products are on the market. Popular approaches include theuse of liposomal formulations, immunoliposomal formulations, variouspolymeric nanotechnologies, antibody-drug fusions/conjugates (ADC),antibody or ligand-protein toxin fusions/conjugates, and dendrimers.Liposomal formulations, including “stealth” approaches are limitedbecause (i) they work only by passive targeting, and (ii) they aredifficult to manufacture on a large scale. Immunoliposomal formulationsovercome the targeting deficiencies of liposomal formulations by addinga targeting component (typically an antibody or antigen binding portionthereof). However, immunoliposomal formulations are even more difficultto manufacture than liposomal formulations, including incorporation of“stealth” technologies. Targeted polymeric nanotechnologies are limitedbecause they require covalent linkage of the payload and targetingmoiety. This complicates manufacturing, limits payload size and variety,and also exposes payload to degradation during circulation in the blood.Antibody-drug fusions/conjugates are limited mostly by payload capacityand payload metabolism. Antibody or ligand-protein toxinfusions/conjugates are also limited by off-target toxicity as well asinsufficient efficacy, primarily due to the inability of the proteintoxin payload to escape the endosomal compartment and effectively reachits cytosolic target following internalization by the target cell.Dendrimers have a larger payload capacity than the antibody-drugfusion/conjugates and can also bind and display targeting moieties,including antibodies. However, dendrimers are also extremely difficultto manufacture because of the complex chemistry and chemicalmanipulation involved in the construction process. Thus, there is a needfor a delivery system to which any antibody (or other targeting moietysuch as a soluble receptor ligand such as VEGF-A) can be bound orcoupled in a simple non-covalent fashion, which can also encapsulatesignificant quantities and combinations of bioactive payloads, and isamenable to large scale manufacturing.

SUMMARY OF THE INVENTION

Some embodiments disclosed herein provide a fully intact bacterialminicell, where the minicell comprises: (i) an Fc binding portion ofProtein G or an Fc binding portion of Protein A displayed on the surfaceof the minicell; (ii) one or more bioactive molecules; and (iii) one ormore Fc-containing targeting molecules bound to said Fc binding portion,wherein said one or more Fc-containing targeting molecules recognize aeukaryotic antigen.

In some embodiments, the minicell comprises an Fc binding portion ofProtein G. In some embodiments, the minicell comprises an Fc bindingportion of Protein A.

In some embodiments, at least one of the one or more bioactive moleculesis a protein toxin. In some embodiments, the protein toxin is selectedfrom the group consisting of gelonin, diphtheria toxin fragment A,diphtheria toxin fragment A/B, tetanus toxin, E. coli heat labile toxin(LTI and/or LTII), cholera toxin, C. perfringes iota toxin, Pseudomonasexotoxin A, shiga toxin, anthrax toxin, MTX (B. sphaericus mosquilicidaltoxin), perfringolysin O, streptolysin, barley toxin, mellitin, anthraxtoxins LF and EF, adenylate cyclase toxin, botulinolysin B,botulinolysin E3, botulinolysin C, botulinum toxin A, cholera toxin,clostridium toxins A, B, and alpha, ricin, shiga A toxin, shiga-like Atoxin, cholera A toxin, pertussis S1 toxin, E. coli heat labile toxin(LTB), pH stable variants of listeriolysin O (pH-independent; amino acidsubstitution L461T), thermostable variants of listeriolysin O (aminoacid substitutions E247M, D320K), pH and thermostable variants oflisteriolysin O (amino acid substitutions E247M, D320K, and L461T),streptolysin O, streptolysin O c, streptolysin O e, sphaericolysin,anthrolysin O, cereolysin, thuringiensilysin O, weihenstephanensilysin,alveolysin, brevilysin, butyriculysin, tetanolysin O, novyilysin,lectinolysin, pneumolysin, mitilysin, pseudopneumolysin, suilysin,intermedilysin, ivanolysin, seeligeriolysin O, vaginolysin, pyolysin,and any combination thereof.

In some embodiments, at least one of the one or more bioactive moleculesis a therapeutic small molecule drug. In some embodiments, thetherapeutic small molecule drug is selected from the group consisting ofDNA damaging agents, agents that inhibit DNA synthesis, microtubule andtubulin binding agents, anti-metabolites, inducers of oxidative damage,anti-angiogenics, endocrine therapies, anti-estrogens, immuno-modulatorssuch as Toll-like receptor agonists or antagonists, histone deacetylaseinhibitors, inhibitors of signal transduction such as inhibitors ofkinases, inhibitors of heat shock proteins, retinoids, inhibitors ofgrowth factor receptors, anti-mitotic compounds, anti-inflammatories,cell cycle regulators, transcription factor inhibitors, and apoptosisinducers, and any combination thereof.

In some embodiments, at least one of the one or more bioactive moleculesis a therapeutic nucleic acid. In some embodiments, at least one of theone or more bioactive molecules is a therapeutic polypeptide. In someembodiments, at least one of the one or more bioactive molecules is acombination of a small molecule drug and a therapeutic nucleic acid.

In some embodiments, at least one of the one or more Fc-containingtargeting molecules is specific for a tumor cell surface molecule. Insome embodiments, at least one of the one or more Fc-containingtargeting molecules is specific for an endothelial cell surfacemolecule. In some embodiments, at least one of the one or moreFc-containing targeting molecules is specific for a target common toboth a tumor cell and an endothelial cell.

In some embodiments, the minicell further comprises an endosomal escapeagent.

Some embodiments enclosed herein provide a composition comprising any ofthe minicells disclosed herein and a pharmaceutically acceptablecarrier.

Some embodiments enclosed herein provide a method of treating a diseasein a subject, where the method comprises administering any of thecompositions disclosed herein to the subject, thereby treating thedisease.

In some embodiments, at least one of the one or more bioactive moleculeis a protein from an infectious agent.

In some embodiments, at least one of the one or more Fc-containingtargeting molecules is specific for a professional antigen presentingcell. In some embodiments, the professional antigen presenting cell is aeukaryotic dendritic cell, eosinophil, neutrophil, basophil, T-cell,B-cell, mast cell, or macrophage.

In some embodiments, at least one of the one or more bioactive moleculesis a protein antigen from a tumor. In some embodiments, at least one ofthe one or more Fc-containing targeting molecules is specific for aeukaryotic dendritic cell or macrophage.

In some embodiments, said minicell further comprises an endosomal escapeagent. In some embodiments, said minicell further comprises animmunomodulatory adjuvant.

Some embodimens disclosed herein provide a method of immunization, wherethe method comprises administering any of the compositions disclosedherein to a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic presentation of an illustrative embodiments of aminicell-based Lateral Flow Immunoassay.

FIG. 2 is a graph showing the level of minicell surface expression anddisplay of the Fc-binding region of either Protein A or Protein Gmeasured by ELISA.

FIG. 3 is a Western Blot showing binding and display of VEGFR2 antibodyto the surface of minciells expressing and displaying the Fc bindingportion of Protein A or Protein G.

FIG. 4 is a Western Blot showing binding and display of EGFR1 antibodyto the surface of minciells expressing and displaying the Fc bindingportion of Protein A or Protein G.

FIG. 5 are images showing fluorescently stained, EGFR1 targetedminicells are internalized by EGFR1-expressing H460 human NSCLC cells inantibody-dependent fashion using Fc-binding minicells expressing anddisplaying Protein G.

FIG. 6A-D are images showing fluorescently stained, EGFR1 targetedminicells are internalized by EGFR1-expressing H460 human NSCLC cells inantibody-dependent fashion using Fc-binding minicells expressing anddisplaying Protein A.

FIG. 7 is a histogram showing relative levels of EGFR1 targeted H460human NSCLC tumor cell internalization of fluorescent minicellsexpressing and displaying Protein A measured by FACS analysis oftrypsinized cells.

DETAILED DESCRIPTION

Definitions

As used herein, the term “Fc-binding minicell” refers to a minicellcomposition in which the minicells and minicell-producing bacterialstrain from which the minicells are derived express and display on theircell surface, a fusion protein comprised of (i) an outer membrane export(secretion) signal, (ii) an outer membrane anchoring domain or anyfunctional equivalent thereof, and (iii) one or more of the Fc bindingdomains of either Protein A or Protein G wherein the minicells arecapable of binding to exogenous antibodies, Fc-containing antibodyderivatives, or Fc-containing fusion/conjugate molecules throughinteraction with the Fc regions of the antibodies and/orfusion/conjugate molecules. In some embodiments, minicells express anddisplay an Fc-binding fusion protein comprised of (i) an outer membraneexport (secretion) signal, (ii) an outer membrane anchoring domain orany functional equivalent thereof, and (iii) the Fc-binding domain of amammalian Fc-receptor (and any functional equivalent thereof) whereinthe minicells are capable of binding to exogenous antibodies orFc-containing fusion/conjugate molecules through interaction with the Fcregions of the antibodies and/or fusion/conjugate molecules.

As used herein, the term “targeted therapeutic minicells” refers tobacterial minicells that encapsulate bioactive molecule(s) of choice,display targeting antibodies and/or other Fc-containing fusion/conjugatetargeting molecules on the external surface of the minicells by way ofinteraction with recombinantly expressed surface localized Fc bindingregions of Protein G or Protein A such that the antibodies and/orFc-containing fusion/conjugate targeting molecules are displayed in sucha way that they are able to specifically bind to, are bound by, or insome other way specifically recognize and thereby deliver, localize to,or aggregate on or within a specific cell, organ, or tissue typeinvolved in the genesis, progression, and/or maintenance of disease, todeliver the molecular contents of said minicell to the target cell,tissue, and organ type in vitro or in vivo. This specific targeting isintended to use minicells to deliver a therapeutic payload to thetargeted cell, organ, and tissue type wherein a therapeutic approach tothe treatment of a disease type listed herein is desirable. The targetedtherapeutic minicells can also contain an endosomal disruption agentincluding but not limited to bacterial cytolysins (such as listeriolysinO (LLO) and perfingolysin O (PFO)) and any functional variants orequivalents thereof. Phospholipases, such as PC-PLC or PI-PLC, can alsobe used as endosomal disrupting agents.

As used herein, the term “targeted diagnostic minicells” refers tobacterial minicells that encapsulate an imaging molecule(s) of choice,displays targeting antibodies and/or Fc-containing fusion/conjugatetargeting molecules on the external surface of the minicells by way ofinteraction with recombinantly expressed surface localized Fc bindingregions of Protein G or Protein A such that the antibodies are displayedin such a way that they are able to specifically bind to, are bound by,or in some other way specifically recognize and thereby deliver,localize to, or aggregate on or within a specific cell, organ, or tissuetype involved in the genesis, progression, and/or maintenance ofdisease, to deliver the molecular imaging contents of the minicell tothe target cell, tissue, and organ type in vitro or in vivo. Thisspecific targeting, in some embodiments, is intended to use minicells toconcentrate molecular imaging agents to the targeted cell, organ, andtissue type wherein a diagnostic approach in whole or in part of adisease type is desirable.

As used herein, the term “targeted minicell vaccine” refers to bacterialminicells that encapsulate a protein antigen and/or a nucleic acid-basedvaccine (e.g. DNA or RNA-based vaccine) derived from an infectiousdisease agent or from a tumor cell of choice, wherein the minicellfurther displays targeting antibodies and/or Fc-containingfusion/conjugate molecules specific for antigen presenting cells of theimmune system on the external surface of the minicells by way ofinteraction with recombinantly expressed surface localized Fc bindingregions of Protein G or Protein A such that antibodies are displayed insuch a way that they are able to specifically bind to, are bound by, orin some other way specifically recognize and thereby deliver, localizeto, or aggregate within antigen presenting cells, organs, or tissuetypes involved in the genesis, progression, and/or maintenance of arecipient host immune response, to deliver the antigenic contents of theminicell to the antigen presenting target cell, tissue, and organ typein vitro or in vivo. Antigen presenting cell-specific targeting, in someembodiments, is intended to use targeted minicell vaccines toconcentrate protein antigen(s) and/or DNA vaccines and/or adjuvant(s) toantigen presenting cells, organs, and tissue types wherein eliciting aprotective recipient host immune response against a particularinfectious or autologous disease type listed herein is desirable. Thetargeted minicell vaccine can include an endosomal disruption agentincluding but not limited to bacterial cytolysins (such as LLO and PFO)and any functional variants or equivalents thereof. Phospholipases, suchas PC-PLC or PI-PLC, can also be used as endosomal disrupting agents.

As used herein, the term “targeting-competent” refers to minicells thatexpress and display one or more Fc binding domains of Protein G orProtein A and are further bound to and display a targeting antibodyand/or Fc-containing fusion/conjugate targeting molecule of interest.

As used herein, the term “Integrin-targeted minicells” refers tominicells that express and display the pan-Beta1-integrin-targeting cellsurface molecule Invasin from Yersinia pseudotuberculosis and anyfunctional equivalents thereof.

As used herein, the term “Integrin-targeted therapeutic minicells”refers to minicells that express and display thepan-Beta1-integrin-targeting cell surface molecule Invasin from Yersiniapseudotuberculosis and any functional equivalents thereof wherein theminicells comprising a bioactive molecule(s) including but not limitedto therapeutic polypeptides, small molecule drugs, therapeutic nucleicacids, and any combination of the preceding. The integrin-targetedtherapeutic minicells can contain an endosomal disruption agentincluding but not limited to bacterial cytolysins (such as LLO and PFO)and any functional variants or equivalents thereof. Phospholipases, suchas PC-PLC or PI-PLC, can also be used as endosomal disrupting agents.

As used herein, the term “cell-specific surface antigen” refers to anyprotein, peptide, carbohydrate or nucleic acid that is preferentiallyexpressed on the surface of or secreted by any tissue, organ or celltype.

As used herein, the term “prokaryotic cell division gene” refers to agene that encodes a gene product that participates in the prokaryoticcell division process. Many cell division genes have been discovered andcharacterized in the art. Examples of cell division genes include, butare not limited to, zipA, sulA, secA, dicA, dicB, dicC, dicF, ftsA,ftsI, ftsN, ftsK, ftsL, ftsQ, ftsW, ftsZ, minC, minD, minE, seqA, ccdB,sfiC, and ddlB.

As used herein, the term “transgene” refers to a gene or geneticmaterial that has been transferred naturally or by any of a number ofgenetic engineering techniques from one organism to another. In someembodiments, the transgene is a segment of DNA containing a genesequence that has been isolated from one organism and is introduced intoa different organism. This non-native segment of DNA may retain theability to produce RNA or protein in the transgenic organism, or it mayalter the normal function of the transgenic organism's genetic code. Insome embodiments, the transgene is an artificially constructed DNAsequence, regardless of whether it contains a gene coding sequence,which is introduced into an organism in which the transgene waspreviously not found.

As used herein, an agent is said to have been “purified” if itsconcentration is increased, and/or the concentration of one or moreundesirable contaminants is decreased, in a composition relative to thecomposition from which the agent has been purified. In some embodiments,purification includes enrichment of an agent in a composition and/orisolation of an agent therefrom.

The term “sufficiently devoid of parental cells”, synonymous with“sufficiently devoid”, as used herein refers to a composition ofpurified minicells that have a parental cell contamination level thathas little or no effect on the toxicity profile and/or therapeuticeffect of targeted therapeutic minicells.

The term “domain” or “protein domain” used herein refers to a region ofa molecule or structure that shares common physical and/or chemicalfeatures. Non-limiting examples of protein domains include hydrophobictransmembrane or peripheral membrane binding regions, globular enzymaticor receptor regions, protein-protein interaction domains, and/or nucleicacid binding domains.

The terms “Eubacteria” and “prokaryote” are used herein as these termsare used by those in the art. The terms “eubacterial” and “prokaryotic”used herein encompass Eubacteria, including both Gram-negative andGram-positive bacteria, prokaryotic viruses (e.g., bacteriophage), andobligate intracellular parasites (e.g., Richettsia, Chlamydia, etc.).

The term “therapeutic nucleic acid” used herein refers to any collectionof diverse nucleic acid molecules that have a therapeutic effect whenintroduced into a eukaryotic organism (e.g., a mammal such as human). Atherapeutic nucleic acid can be a ssDNA, a dsDNA, a ssRNA (including ashRNA), a dsRNA (including siRNA), a tRNA (including a rare codon usagetRNA), a mRNA, a micro RNA (miRNA), a ribosomal RNA (rRNA), a peptidenucleic acid (PNA), a DNA:RNA hybrid, an antisense oligonucleotide, aribozyme, an aptamer, or any combination thereof.

The term “therapeutic polypeptide” used herein refers to any collectionof diverse protein molecule types that have a therapeutic effect whenintroduced into a eukaryotic organism (e.g., a mammal such as human). Atherapeutic polypeptide can be a protein toxin, a cholesterol-dependentcytolysin, a functional enzyme, an activated caspase, a pro-caspase, acytokine, a chemokine, a cell-penetrating peptide, or any combinationand/or plurality of the proceeding.

The term “overexpression” used herein refers to the expression of afunctional nucleic acid, polypeptide or protein encoded by DNA in a hostcell, wherein the nucleic acid, polypeptide or protein is either notnormally present in the host cell, or wherein the nucleic acid,polypeptide or protein is present in the host cell at a higher levelthan that normally expressed from the endogenous gene encoding thenucleic acid, polypeptide or protein.

The term “modulate” as used herein means to interact with a targeteither directly or indirectly so as to alter the activity of the targetto regulate a biological process. The mode of “modulate” includes, butis not limited to, enhancing the activity of the target, inhibiting theactivity of the target, limiting the activity of the target, andextending the activity of the target.

The term “heterologous” as used herein refers to a protein, gene,nucleic acid, imaging agent, buffer component, or any other biologicallyactive or inactive material that is not naturally found in a minicell orminicell-producing bacterial strain and is expressed, transcribed,translated, amplified or otherwise generated by minicell-producingbacterial strains that harbor recombinant genetic material coding forsaid heterologous material or coding for genes that are capable ofproducing said heterologous material (e.g., a bioactive metabolite notnative to the parent cell).

The term “exogenous” as used herein refers to a protein (includingantibodies), gene, nucleic acid, small molecule drug, imaging agent,buffer, radionuclide, or any other biologically active or inactivematerial that is not native to a cell, or in the case of a minicell, notnative to the parent cell of the minicell. Exogenous material differsfrom heterologous material by virtue of the fact that it is generated,purified, and added separately.

The term “therapeutic” as used herein means having a biological effector combination of biological effects that prevents, inhibits,eliminates, or prevents progression of a disease or other aberrantbiological processes in an animal.

The term “diagnostic” as used herein means having the ability to detect,monitor, follow, and/or identify a disease or condition in an animal(including humans) or from a biological sample including but not limitedto blood, urine, saliva, sweat and fecal matters.

The term “theranostic” as used herein means having the combined effectsof a therapeutic and a diagnostic composition.

The term “recombinantly expressed” as used herein means the expressionof one or more nucleic acid(s) and/or protein(s) from a nucleic acidmolecule that is artificially constructed using modern geneticengineering techniques wherein the artificially constructed nucleic acidmolecule does not occur naturally in minicells and/or minicell-producingbacterial strains wherein the artificial nucleic acid molecule ispresent as an episomal nucleic acid molecule or as part of theminicell-producing bacterial chromosome.

The term “episomal” as used herein means a nucleic acid molecule that isindependent of the chromosome(s) of a given organism or cell.

The term “detoxified” as used herein refers to a modification made to acomposition or component thereof that results in a significant reductionin acute toxicity to the modified composition or component thereof,regardless of what the causative biological basis for toxicity to thecomposition or component thereof happens to be.

The term “gene silencing” as used herein refers to a specific reductionof the intracellular pool of mRNA for a given protein compared to thenormal level of the mRNA as a result of the delivery of a therapeuticnucleic acid delivered by targeted minicells. The therapeutic nucleicacids include, but are not limited to, double stranded RNAs (e.g.,siRNA) as well as single stranded RNAs (e.g., shRNA and miRNA) and anyeukaryotic expression plasmids encoding the same.

The term “eukaryotic expression plasmid” as used herein refers to acircular double stranded DNA molecule that encodes for one or more geneproducts operably linked to eukaryotic expression control sequences suchthat the gene product(s) can be transcribed and translated by aeukaryotic cell from the double stranded DNA molecule.

As used herein, the term “bioactive molecule” refers to a moleculehaving a biological effect on an eukaryotic organism (e.g., a mammalsuch as human) when introduced into the eukaryotic organism or cell.Bioactive molecules include, but are not limited to, therapeutic nucleicacids, therapeutic polypeptides (including protein toxins), andtherapeutic small molecule drugs.

As used herein, the term “Fc-containing targeting molecule” refers to amolecule that is capable of binding to an Fc binding molecule (e.g., theFc binding portion of Protein A or Protein G) and contains a recognitionsite for a target molecule (e.g., an antigen or a receptor).Fc-containing targeting molecules include, but are not limited to,antibodies having an Fc region and soluble receptor ligands engineeredto contain an Fc region.

As used herein, the term “eukaryotic antigen” refers to an antigen thatof an eukaryotic origin, for example, an antigen displayed on thesurface of a eukaryotic cell.

As used herein, the term “protein toxin” refers to a protein that has atoxic effect on eukaryotic cells.

As used herein, the term “small molecule” refers to a molecule that hasa biological effect and that has a molecular weight of less than 5000Daltons. In some embodiments, small molecules have a molecular weight ofless than 2500 Daltons. In some embodiments, small molecules have amolecular weight of less than 1000 Daltons. In some embodiments, smallmolecules have a molecular weight of less than 800 Daltons. In someembodiments, small molecules have a molecular weight of less than 500Daltons.

As used herein, the term “therapeutic small molecule drug” or “smallmolecule drug” refers to a small molecule that has a therapeutic effectwhen introduced into a eukaryotic organism (e.g., a mammal such ashuman).

Description

The present application relates to the use of bacterial minicells as invitro and in vivo targeted bioactive molecule delivery and vaccineagents. Eubacterial minicells have a distinct advantage as deliveryvehicles, in that they can be engineered to target and deliver largenumbers and a large variety of bioactive molecules to specific celltypes in vivo. Bacterial minicells are designed to display antibodiesand/or other Fc-containing fusions/conjugates on their surfaces thatspecifically target the minicell to cell types or tissues involved inthe initiation, promotion, support, and maintenance of disease or otheraberrant biological processes in an animal.

Minicells are achromosomal, membrane-encapsulated biologicalnanoparticles (approximately 250-500 nm in diameter) that are formed bybacteria following a disruption in the normal division apparatus ofbacterial cells. In essence, minicells are small, metabolically activereplicas of normal bacterial cells with the exception that they containno chromosomal DNA and as such, are non-dividing and non-viable.Although minicells do not contain bacterial chromosomes, plasmid DNAmolecules (smaller than chromosomes), RNA molecules (of all subtypes),native and/or recombinantly expressed proteins, and other metaboliteshave all been shown to segregate into minicells. Minicells are uniquelysuited as in vivo therapeutic delivery, diagnostic, theranostic, andimaging vehicles because they combine many of the individual advantagesof other delivery technologies into a single, versatile deliveryvehicle. Minicells can be “engineered” to preferentially encapsulate, becoupled to, or absorb biologically active molecules, including variousnucleic acids, proteins, small molecule drugs, and any combinationthereof for subsequent delivery in both prophylactic and therapeuticmedicinal applications where the detection, prevention, maintenance,and/or inhibition of disease is desirable. As described herein,minicells have the advantage that they can be engineered to selectivelytarget specific cell types responsible for disease through the use of anovel surface display system capable of displaying any Fcregion-containing antibody or Fc-region containing antibody derivative,as well as Fc region-containing fusions or conjugates that include butare not limited to polypeptides, nucleic acids, DARPins, radionuclides,carbohydrates, small molecules, and imaging agents.

Another advantage of the use of minicells as delivery vehicles(regardless if they are targeted or non-targeted) is that bioactivemolecules can be delivered in combination as described by U.S. Pat. No.7,183,105, which is incorporated herein by reference in its entirety.For example, it has been demonstrated that minicells can successfullygenerate humoral immune responses against a heterologous antigen whenused as a delivery vehicle for plasmid DNA vaccines. When minicells areused to simultaneously deliver both a DNA vaccine and the correspondingprotein antigen, humoral responses were greatly improved, illustratingthe benefits of the flexibility of minicells with respect to deliveryoptions. As described herein, minicells have unique features that allowfor the loading of small molecule drug and imaging agents as well as thedistinct ability to recombinantly express and encapsulate therapeuticnucleic acid delivery molecules, peptides, and proteins for delivery.These unique features allow for a highly flexible delivery system thatcan deliver multiple payloads of different molecular origins in concert.

Approaches for targeting minicells to eukaryotic cells in vitro and invivo include (i) random chemical coupling of antibodies, antibodyfragments, or other antibody derivates to the surfaces of minicellsusing myriad chemical coupling techniques known in the art, (ii) usingbi-specific antibodies, bi-specific antibody fragments, or bi-specificantibody derivatives to non-covalently attach targeting antibodies tothe surfaces of minicells, and (iii) expressing a single chain antibodyor other antibody fragment on the surface of the minicells in thecontext of a contiguous fusion with a minicell membrane-anchoringprotein such as a bacterial outer membrane protein. The presentapplication describes a novel approach that provides significantadvantages with respect to manufacturing, immunogenicity, and targetingof therapeutic minicells.

In instances where exogenous antibodies existing free in solution arecross-linked to the surfaces of minicells, there is a lack of controlover orientation of the antibody or Fc-fusion/conjugate because thesemolecules will be randomly cross-linked to the surface of minicells inmany different orientations. This has two potential effects on the endproduct that would add to the manufacturing complexity and have thepotential to diminish efficacy. The first deleterious effect onlyapplies to the cross-linking of full length antibodies and its exposureof the Fc regions instead of the binding regions, depending on which endof the antibody or other Fc-region containing fusion/conjugate becomescoupled to the minicell surface. The Fc-region has the potential tostimulate the immune system by virtue of the interaction of theFc-region with various components of the immune system (e.g., the Fcreceptor on the surface of Natural Killer cells). The second deleteriouseffect is that there will be an inherent heterogeneity with respect tothe orientation of the antibody (or other Fc-region containingfusion/conjugate) on the surface, such that not all binding regions areexposed. Variable binding portion exposure has the potential to resultin diminished and/or variable efficacy as a result of fewer functionalbinding moieties on the surface of the minicell. Either or both of theselimitations have the potential to increase the immunogenicity and/orclearance of minicells, making them less effective therapeutic deliveryvehicles. However, when used in the context of the present disclosure,chemical cross-linking of antibodies to the surface of minicellscircumvents the issues described above because the Fc-binding minicellsof the present disclosure help to orientate the antibodies such that (i)Fc regions of the antibodies are concealed and (ii) the antigen bindingsites of bound and cross-linked antibodies are optimally displayed (i.e.pointed outward from the minicell versus a randomized orientation). Asused herein, cross-linking reagents can be “homobifunctional” or“heterobifunctional” (having the same or different reactive groups,respectively). Examples of cross-linking reagents include, but are notlimited to, those listed in Table 1. In this context, a preferred methodwith respect to cross-linking include generating and purifyingFc-binding minicells as described herein, incubating the minicells in asolution containing the targeting antibodies of choice, allowing bindingto occur, washing excess unbound antibody away, performing thecross-linking reaction, and subsequently removing excess cross-linkingreagent using standard methods known in the art.

The compositions and methods disclosed herein are advantageous over someof the compositions and methods where bi-specific antibodies,bi-specific antibody fragments, and bi-specific antibody derivatives areused. For example, it can be costly to make bi-specific antibodies thathave specificity for a native minicell surface component on one antibodyarm and specificity for a eukaryotic cell surface target on the other.In addition, bi-specific antibodies, much like antibodies chemicallyconjugated to the surface of minicells, would expose the Fc region,potentially activating complement and/or making the Fc region accessibleto Fc-binding cells of the immune system in vivo. Bi-specific antibodyfragments that do not include the Fc region can circumvent the issuesrelated to Fc region exposure in vivo, except that these types ofmolecules require even more genetic engineering than that of abi-specific antibody. In the case of the construction of bi-specificantibody complexes, there are multiple drawbacks. If the bispecificantibody complex is made using covalent cross-linking methods known inthe art, the same limitations apply as in the case where antibodies arecross-linked directly to minicells with respect to having to purify awayexcess chemical cross-linking agent. As bio-specific antibody complexesare made by mixing equimolar amounts of antibody together followed bythe addition of exogenous chemical cross-linking agent to catalyze thereaction, further purification to remove the undesired duallymono-specific species is required. Failure to remove undesired duallymono-specific species would result in crosslinking of minicells to otherminicells as a result of the presence of mono-specific antibodies withaffinities for minicells. In a related approach obviated by theteachings of U.S. Pat. No. 7,183,105, a hybrid Protein A/G molecule isused as a non-covalent scaffold by which to link two differentmono-specific antibodies together to form a “bi-specific ligand” asdescribed in U.S. Pat. Nos. 8,772,013, 8,691,963 and 9,169,495, each ofwhich is hereby incorporated by reference in its entirety. Theaggregation problem is amplified with the use of this approach becausethe Protein A/G molecule used as the scaffold indiscriminately binds six(6) different antibodies per Protein A/G molecule. Again, two separateantibody types are mixed together in equimolar amounts followed by theaddition of Protein A/G to non-covalently bind and link the twodifferent antibodies together. In this approach, 720 differentpermutations of antibody complexes are made, most of which have affinityfor two (2) to six (6) minicells. This limitation requires costly andcomplex manufacturing procedures to be put in place in order to complywith GMP standards. This is in addition to the cost of all of thedifferent components required to manufacture this type of minicellproduct. An additional problem is the significant potential for toxicityassociated with administration of an aggregated product to a patient. Aswith chemical cross-linking, these limitations also have the potentialto increase the immunogenicity and/or clearance of minicells, makingthem less effective therapeutic delivery vehicles. The Fc-bindingminicells disclosed herein reduce immunogenicity when the antibody isderived from the species to which they are administered as a treatmentmodality. Because Fc-binding minicells bind the Fc regions of antibodiesor Fc-containing fusions/conjugates, the Fc regions are thereby maskedfrom the Fc receptor expressing cells of the immune system (e.g.,macrophages and NK cells). Further, when the antibody utilized isderived from the species from which the minicell is to be administeredas a treatment modality, the minicells become “stealthy” in that thesurface is now covered by “self' proteins (antibodies). Immunocompetentorganisms do not readily recognize “self' proteins. Minicells that havebound to and display “self' proteins (e.g. antibodies) are therebyfurther masked from the immune system. Masking targeted therapeuticminicells from the immune system is advantageous because it can increasethe in vivo half-life of the minicells providing a longer window for theminicells to reach their intended target.

In the case of the expression and display of a single chain antibody onthe surface of the minicell in the context of a contiguous fusionprotein to the extracellular domain of a bacterial outer membraneprotein, there are two limitations. The first is that not everymonoclonal antibody sequence can be converted into a single chainantibody fragment and maintain the same binding properties as theoriginal parent monoclonal antibody molecule. The second limitation isthat even in instances where a monoclonal antibody can be converted intoa single chain antibody fragment, binding capability sometimes has to beoptimized via generation of a variety of fusion sequences and linkerconstructs. Thus, the single chain antibody display approach is limitedonly to single chain antibodies that maintain the activity, in whole orin large part, of the parent antibody molecule. While many single chainantibodies exist and can be incorporated into minicell compositions thatexpress and display single chain antibodies or antibody fragments, it isstill advantageous to be able to display full length monoclonalantibodies, as taught here, because it further expands the repertoire ofantibodies from which the artisan may choose and significantly speeds upthe process of selecting potentially successful drug developmentcandidates. In some embodiments, the Fc-binding minicells disclosedherein can be used to “screen” a library of exogenous whole antibodiesto select for antibody candidates useful when converted to a singlechain for expression and display on the surface of minicells. In otherembodiments, the Fc-binding minicells disclosed herein can be employedto screen a library of single chain antibodies as a primary selectionprocess for making determinations as to which single chain antibodieswill maintain their binding and internalization properties if convertedto a fusion protein designed to be expressed and displayed on thesurface of minicells. In yet another example, Fc-binding minicells canbe used to screen Fc-containing fusion or conjugated proteins. SuchFc-containing fusions and conjugates are described in more detailherein.

A novel approach for overcoming many of the limitations described aboveis disclosed herein, in which antibodies or other Fc-region containingfusions/conjugates are non-covalently coupled directly to the surface ofminicells that express and display a fusion protein that is comprised of(i) an outer membrane export (secretion) sequence, (ii) an outermembrane protein or membrane anchoring portion thereof, and (iii) the Fcbinding portion(s) of Protein A or Protein G on the minicell surface.Minicells displaying one or more of the Fc binding region(s) of ProteinA or Protein G can bind full length antibodies and/or other Fc-regioncontaining fusions/conjugates through the Fc region of the antibodies orfusions/conjugates, with no modification or manipulation of theminicells, antibodies, or Fc-region containing fusions/conjugates by wayof co-incubation of the minicells with the antibodies or Fc-regioncontaining fusions/conjugates.

The compositions and methods disclosed herein are advantageous over thecoupling approaches listed above, for example, they are designed todisplay antibodies or other Fc-containing fusions/conjugates such that(i) the Fc region of the antibody is concealed and (ii) the antigenbinding/effector domain of the antibodies and/or fusions/conjugates areoptimally exposed by virtue of the binding of the antibody orFc-containing fusion/conjugate to the Protein A or Protein G Fc-bindingfusion molecule on the surface of the minicell. The minicells can thenbe further loaded with one or more species of bioactive payload(s)either by way of exogenous addition of the payload to purified minicellsor by recombinant expression of the payload from the minicell-producingparent bacterium prior to or during minicell formation. Bioactivepayloads that are expressed or loaded into minicells include but are notlimited to small molecule drugs and/or a radionuclide, a therapeuticsingle stranded short hairpin RNA (ssRNA, a.k.a. shRNA), a therapeuticdouble stranded RNA molecule (e.g., a siRNA), a ribozyme, an aptamer, atherapeutic polypeptide (e.g., a protein toxin), a eukaryotic expressionplasmid encoding for a therapeutic polypeptide or therapeutic nucleicacid, and any combination of the preceding bioactive payloads. In someembodiments, minicells loaded with a bioactive payload or combinationthereof and also displaying antibodies or other fusions/conjugates thatrecognize eukaryotic cell surface molecules are comprised of mutantde-toxified lipopolysaccharide (LPS) molecules as described herein.

In some embodiments, targeted minicells comprising bioactive payload(s)target and engage their cognate eukaryotic cell surface molecule in vivoor in vitro, stimulate endocytosis of the minicell(s), and are degraded,thereby releasing their contents directly into the targeted eukaryoticcell. In some other embodiments, minicells comprising bioactivepayload(s) target and engage their cognate eukaryotic cell surfacemolecule in vivo or in vitro but do not stimulate endocytosis of theminicell(s). These are termed “localized” minicells and are eventuallydegraded at or near the target cell surface, thereby releasing theircontents directly in the vicinity of the targeted eukaryotic cellwhereby the payload can exert its therapeutic effect on the target cell.The minicells and minicell-producing bacterial strains disclosed hereinhave been genetically engineered to express and display one or more ofthe Fc binding portions of Protein G or Protein A such that they arecapable of recognizing and binding to the Fc region of antibodies and/orother Fc-containing fusions/conjugates. Antibodies and/or Fc-containingfusions/conjugates bound by minicells displaying one or more of the Fcbinding regions of Protein G or Protein A constitute the targetingcomponent of the targeted minicells disclosed herein.

Protein G is a cell-surface protein expressed by the Gram-positivebacterium Group G Streptococcus. Its natural biological function is toprevent opsonization of Group G Streptococcus during the infectionprocess by binding the Fc region of antibodies such that the Fc regionis masked from the immune system. Normally, anti-bacterial cell surfaceantigen antibodies bind to the surface of bacterial cells and induceopsonization and/or the activation of complement depending on externalexposure of the Fc regions of the bacterial cell surface-boundantibodies. Protein G serves to prevent this in Group G Streptococcus bybinding the Fc region of antibodies, thereby masking the exposed Fcregion from the immune system. Protein G is a 51 kilodalton proteinconsisting of 13 distinct domains that are commonly further consolidatedto include the A/B domains, the S domain, the C/D domains, and the W/Mdomain. The A/B domains of Protein G consist if three highly relatedrepeats (A1-B1; A2-B2; A3) that have overlapping binding sites for theFab region of antibodies (moderate affinity) and for serum albumin (highaffinity). The S domain is the spacer domain between the A/B and C/Ddomains. The C/D domain consists of three more highly related repeats(C1-D1; C2-D2; D3) and constitutes the Fc binding region of themolecule. The C/D domain is capable of binding two (2) separateantibodies by the antibody Fc region. Thus, Protein G contains two Fcbinding domains, either or both of which can be utilized in theembodiments disclosed herein. The C/D region is commonly used toaffinity purify antibodies from serum (or other sources). The W/Mdomains function to interact with the Gram-positive cell wall and tofacilitate export to the outer leaflet of the cell membrane,respectively. The Protein G fusion protein(s) disclosed herein thereforeinclude, but are not limited to, the S and C/D regions of Protein G. Insome embodiments, it is advantageous to include only the S and C/Dregions in the design of the recombinant fusion proteins disclosedherein to avoid unwanted binding of the Fab regions of antibodies(exposes the Fc region) or to serum albumin. In some embodiments, theserum albumin binding domain of Protein G is included such thatminicells can be made into “enhanced stealth minicells” by way ofbinding serum albumin to the surface of the minicell in addition to anantibody. Minicell surface-bound serum albumin, in addition to theantibody, helps to mask the minicell from the recipient immune system.In these cases, it is preferred to match both species of origin of boththe antibody and the serum albumin with that of the recipient.

Protein A is a cell-surface protein expressed by the Gram-positivebacterium Staphylococcus aureus. Like Protein G, its natural biologicalfunction is also to prevent opsonization of Staphylococcus aureus duringthe infection process. Staphylococcus aureus use Protein A to bind tothe Fc region of antibodies. Depending on orientation or externalexposure, the Fc regions of surface-bound antibodies are capable ofactivating complement or binding to Fc receptors on phagocytic cellssuch as neutrophils. Protein A is a 58 kilodalton protein that consistsof 7 distinct domains that are commonly referred to as the S, E, D, A,B, C, and X domains. The S domain constitutes the secretion signal. TheE domain is not well characterized and has no immunoglobulin bindingactivity. Domains D, A, B, and C, often grouped together and referred toas the Z domain, contain four (4) consecutive immunoglobulin bindingdomains, thought to have evolved as a result of gene duplications in S.aureus. The D, A, B, and C domains may be uncoupled and each maintainsits immunoglobulin binding properties with little or no effect onaffinity/specificity. Protein A contains four discreet Fc bindingdomains, any of which can be utilized in singular or in combinationherein. Thus, different Protein A derived Fc-binding fusion proteins cancontain 1, 2, 3, or 4 Fc-binding domains and can be incorporated in thecompositions and methods disclosed herein at the discretion of a skilledartisan. The X domain has no immunoglobulin binding activity and isresponsible for anchoring the C-terminus back into the peptidoglycanwall in the Gram-positive setting. The X domain is dispensable and hasno effect on the binding properties of the D, A, B, and C domains. TheProtein A fusion protein(s) disclosed herein therefore include, but arenot limited to, the D, A, B, and C domains of Protein A or derivativesof one or more of these domains that retain Fc binding capability.Preferred derivatives include domains in which glycine 29 is substitutedfor alanine, eliminating F(ab′)₂ binding, and/or derivatives in whichputative cleavage sites by OmpT protease have been eliminated bysubstitution with functionally conserved amino acid residues.

Fc-binding minicells expressing either the Fc binding region of ProteinG or that of Protein A can be used to properly display antibodies and/orother Fc-containing fusions/conjugates on the surfaces of minicellswhich serves to facilitate targeting of minicells to specific cell,tissue, and organ types in vivo. Antibodies, or any Fc containingportion thereof, intended to aid in the targeting of minicells to aspecific tissue, organ, and cell type involved in the cause,progression, maintenance, or manifestation of disease can be derivedfrom or be part of any immunoglobulin or immunoglobulin subclass,including but not limited to IgA, IgM, IgD, IgG, and IgE. Antibodies ofany subclass intended to facilitate the targeting function of minicellscan be “humanized”, although any antibody of any subclass against a cellspecific antigen can be raised in any animal known to generate antibodyresponses through adaptive immunity to achieve the same goal. In nature,antibodies are generated such that they contain two separate arms(Fab's), each of which recognizes the same epitope of a particularantigen. However, as described below, advances in molecular biology haveenabled researchers to modify the specificity of each arm (or in somecases the Fc region of the molecule) to recognize distinctly differentepitopes that may or may not occur in the same or different antigens.These antibody derivatives are referred to as a ‘bispecific’ antibodiesor ‘bispecific’ targeting moieties.

In the laboratory, antibodies can be engineered to be independentlyspecific for different antigens, such that a single antibody targets twoseparate antigens simultaneously. By way of non-limiting example,antibodies can be engineered to recognize putative surface components ofa given eubacterial minicell (e.g., LPS O-antigens) on one Fab′ and theother Fab′ of the bispecific antibody can be engineered to recognize aeukaryotic cell-specific surface antigen. In another non-limitingvariation on this theme, the Fc region of the heavy chains of theantibody can be genetically engineered to specifically bind to aparticular epitope within a given antigen (e.g., LPS) while the Fab′portions of the molecule recognize a different epitope in a separateeukaryotic cell-specific surface antigen or vice versa.

Additionally, those skilled in the art will readily recognize that twoseparate antibodies, with separate specificities, can be non-covalentlyattached by coupling them to soluble Protein A, Protein G, or ProteinA/G (or any other binding molecule that will recognize and bind two ormore antibodies) to form a bispecific antibody derivative capable ofadhering to the surface of minicells wherein one antibody within thecomplex specifically adheres to the surface of the minicell and theother antibody is displayed to specifically recognize and thereby“target” a specific cell, tissue, or organ type expressing an eukaryoticcell-specific surface antigen in vivo. Similarly, one skilled in the artwill recognize that two separate antibodies, with separatespecificities, can be covalently linked using myriad cross-linkingtechniques to achieve the same effect.

Other, non-antibody based targeting approaches disclosed in the presentapplication are collectively based on Fc-containing fusions orconjugates. As described herein, examples of molecular targetingmoieties includes, but are not limited to, receptor ligands,polypeptides, hormones, carbohydrates, aptamers, antibody-likemolecules, nanobodies, affibodies, antibody-like single chain T-cellantigen receptors (STARs), mTCRs, trans-bodies, XmAbs, and DARPins.Fc-conjugation can be achieved using a variety of approaches known inthe art. By way of non-limiting example, the soluble EGF or VEGF ligandscan be genetically fused or conjugated to an Fc-containing polypeptide(Fc region) and bound to the Fc-binding minicell surface such that theFc-binding minicells are targeting competent and can selectivelylocalize and be internalized by cells expressing the EGFR or VEGFR2receptor, respectively. As another non-limiting example, the therapeuticpayload itself can be genetically fused or coupled to an Fc-containingpolypeptide and bound to the surface of the Fc-binding minicells. Forexample, Fc-conjugated siRNA molecules can be bound to the surface ofFc-binding minicells in addition to Fc-containing antibodies,Fc-containing antibody derivatives, and/or Fc-containingfusion/conjugate targeting molecules. Fc-containing polypeptide fusionsinclude, but are not limited to, receptor ligand/Fc fusions,Fc-containing peptide fusions, and Fc-containing DARPins. Recombinantexpression of the fusion is a preferred method of construction. In therecombinant expression context, Fc regions can be fused to either theamino or carboxy terminus of a given recombinant fusion at thediscretion of the skilled artisans such that fusion to the Fc regiondoes not affect ligand activity with respect to receptor binding andstimulation of receptor-mediated endocytosis. Another exemplary approachto making Fc-containing polypeptides, peptides, and DARPins is bychemical conjugation (a.k.a. cross-linking) of purified recombinant Fcregion molecules to recombinant polypeptide, peptide, and/or DARPinmolecules using any of the cross-linking techniques known in the art. Inthe context of chemical cross-linking, it is advantageous to include“reactive” amino acid groups on either or both of the purifiedrecombinant Fc-region or the polypeptide, peptide, and/or DARPinmolecule to be conjugated. Examples of reactive amino acids include, butare not limited to, those that contain sulfhydryl groups, preferably acysteine residue. In some embodiments, for use with popularheterobifunctional cross-linking reagents, it is preferable to include alysine residue at the linkage site of the opposing conjugate (e.g.,Fc-region contains a cysteine residue while targeting or payloadpolypeptide contains a lysine or vice versa). In instances wherepurified recombinant Fc regions are cross-linked to hormones,carbohydrates, aptamers, and other non-amino acid and/or peptide basedmolecules, the skilled artisans would recognize that many othercross-linking reagents can be employed to achieve the same.Cross-linking reagents can be “homobifunctional” or “heterobifunctional”(having the same or different reactive groups, respectively). Examplesof cross-linking reagents include, but are not limited to, those listedin Table 1. Table 1 also illustrates non-limiting examples ofcross-linking reagents that can be used for each conjugate moleculetype/approach.

In some preferred embodiments, minicells and minicell-producingbacterial strains are “engineered” to express and display a recombinantFc binding portion of Protein G or Protein A on their surfaces. Surfacelocalization of recombinant polypeptides has been successfullyaccomplished in Salmonella enterica by using fusion proteins thatcontain an Antigen 43-α outer membrane anchoring domain fused to asingle chain FcV antibody fragment with specificity for Chlam 12 orCTP3. In a similar study, E. coli cells expressing and displaying singlechain FcV antibody fragments directed towards Coronavirus epitopes fusedwith the outer membrane localized IgA protease of Neisseria gonnorhoeaewere shown to neutralize Coronavirus and prevent infection in vitro.Surface localization can also be accomplished by fusing coding sequencesof the desired Fc binding protein with theadhesin-involved-in-diffuse-adherance (AIDA-I) autotransporter from E.coli. This can also be accomplished with the Lpp-OmpA whole cell displaysystem described in U.S. Pat. No. 5,348,867, which is incorporatedherein by reference. In some embodiments, Lpp-OmpA is used to expressand display antibody Fc binding moieties including but not limited tothe Fc binding region of Protein A or Protein G on the surfaces ofminicells. Other native outer membrane proteins that can serve as theouter membrane fusion partner include, but are not limited to, LamB,OmpF, OmpC, OmpD, PhoE, PAL, pilus proteins, and various flagellins ingram negative Enterobacteriacea family members. This approach is used toexpress and display Fc-binding fragments of Protein A or Protein G onthe surface of minicells derived from any Enterobacteriacea orBacillaceae family member such that the minicells are capable of bindingto and displaying antibodies against cell surface antigens therebybecoming specific targeted delivery vehicles for cell surfaceantigen-expressing cells, tissues, or organs. One skilled in the artwill recognize that achieving this goal is a matter of (i) creating anucleic acid sequence encoding for a fusion protein between a putativeor predicted outer membrane protein or outer membrane localizationsequence and the Fc binding domain(s) of Protein A or Protein G, (ii)producing and purifying minicells that express the fusion protein(a.k.a. Fc-binding minicells), (iii) loading minicells with a smallmolecule drug or other bioactive payload (not required when minicellsencapsulate a recombinantly expressed payload or payload combinations;e.g., a protein toxin or a protein toxin and an shRNA combination), (iv)incubating payload-loaded minicells with the targeting antibody or otherFc-containing fusion/conjugate to make targeted therapeutic minicells,(v) washing the preparation to remove excess payload (where applicable)and antibody or Fc-containing fusion/conjugate molecules, and (vi)preparing the minicells as a pharmaceutical composition per the intendedroute of patient administration.

Bacterial minicells have distinct small molecule drug and imaging agentloading advantages over other delivery technologies. Similar tode-energized bacterial cells, targeted minicells can be easily loadedwith high concentrations of small molecule drugs and imaging agents bysimple co-incubation of purified minicells with a high concentration ofthe small molecule drug or imaging agent. Optimally, the small moleculedrug(s) and/or imaging agent(s) are incubated with minicells in aloading buffer that is devoid of any exogenous energy source so as tomaintain the inactive state of conserved multi-drug efflux pumps.Multi-drug efflux pumps are largely proton motive force (PMF) dependentand it is well recognized by the skilled artisan that the PMF andthereby the efflux pumps are dependent upon an exogenous energy source.Thus, loading minicells in an energy source-free buffer ensures theinactivity of the efflux pump system(s) of minicells and serves todiminish drug efflux from minicells even when drug-loaded minicells arerestored to a medium that reverses the concentration gradient of thedrug (i.e., drugless medium). Targeted minicells can also be used todeliver two or more small molecule drugs or imaging agentssimultaneously such that several intracellular targets are addressed ina single delivery event. In addition, targeted minicells can also beused to deliver one or more small molecule drugs in concert with one ormore therapeutic nucleic acids.

Effective delivery of small molecules by way of receptor mediatedendocytosis can be limited if the small molecule(s) delivered areexposed to the harsh environment of the endosomal or lysosomalcompartments for too long prior to being released to the cytosol of thetargeted eukaryotic cell. Thus, the skilled artisans will appreciatethat enhanced endosomal escape of small molecules delivered by targetedminicells by this route may be desirable. However, this is not alwaysnecessary and can be subjected to the discretion of the skilled artisansand may also be employed in the delivery of other targetedminicell-borne payloads including but not limited to nucleic acids,peptides, proteins, and radionucleotides and any combination of thepreceding. Intracellular pathogens are faced with the same problem andas a result have evolved sophisticated mechanisms to either modulate theenvironment of the endosome to make it hospitable or to escape theendosome completely. In the case of the latter, this is typicallymediated by a protein component or protein complex made by the invadingorganism. For example, the listeriolysin O (LLO; SEQ ID NO:29) proteinof the intracellular Gram-positive pathogenic bacterium Listeriamonocytogenes can be used in the methods and compositions disclosedherein. Listeriolysin O is a 58 kilodalton secreted pH andcholesterol-dependent protein encoded by the hlyA gene of Listeriamonocytogenes that forms oligomeric pores in the endosomal membrane,facilitating the escape of the invading organism into the cytosol of theinfected cell. In some embodiments, full length LLO (containing thesignal secretion sequence) is used as the endosomal membrane disruptionagent. As skilled artisans will appreciate that other useful variants ofLLO that have been described can also be used in the presentapplication. For example, in nature, the secretion of LLO by Listeriamonocytogenes (and other Gram-positive bacterial species) involves thecleavage of the 24 amino acid signal secretion sequence by membraneproteases to form “mature” LLO. Removing the 24 amino acid-longsecretion signal from LLO using recombinant methods results in thesequestration of truncated LLO (cLLO; SEQ ID NO 30) in the bacterialcytosol when expressed. Although cLLO is not secreted, it maintains allof the properties of the secreted form, including its pH andcholesterol-dependent endosomal membrane pore forming capabilities. Insome embodiments, the signal sequence of LLO is removed at the geneticlevel using recombinant techniques known in the art. It has been shownthat LLO is a heat labile protein that undergoes an irreversibleconformational change that abrogates activity at neutral pH andtemperatures above 30° C. Thermostability of LLO can be increased when acombination of amino acid substitutions are made (E247M and D320K; SEQID NO 31). In some embodiments, a thermostable version of LLO is used.Upon targeting of the minicell(s), receptor mediated endocytosis carriesthe minicell into the endosome. The harsh environment of the endosomebegins to degrade the engulfed minicell, co-releasing the small moleculepayload along with LLO. The released LLO component then aids tofacilitate release of the small molecule from the endosome into thecytosol where the small molecule can exert its biological effect(s). Asdisclosed herein, certain temperature and degradation-stabilized (sLLO)and pH stabilized (pH-independent; sLLOpH) variants of LLO (SEQ IDNO:32) can serve as a therapeutic polypeptide payload as well as anendosomal disruption agent. As used herein, the listeriolysin O (LLO)protein include the full length LLO as well as the truncated LLO (cLLO).Various mutations have been reported, which modulate the cytotoxicity ofPFO, without significantly compromising endosomal disruption activity.PFO and said mutational variants can be used in place of LLO for thepurpose of endosomal membrane disruption.

Minicells have distinct mechanisms and advantages of loading therapeuticnucleic acids and therapeutic polypeptides (e.g. protein toxins) asopposed to other targeted delivery technologies. For example, theminicell-producing parental cells can be used to recombinantlyexpress/produce one or more therapeutic nucleic acid molecules and/ortherapeutic polypeptides prior to or at the same time that minicells arebeing produced. Recombinant therapeutic nucleic acids and/or therapeuticpolypeptides are expressed, segregate into and are encapsulated byminicells, and are then delivered to eukaryotic cells by targetedminicells in vivo or in vitro.

Examples of recombinantly expressed/produced therapeutic nucleic acidsto be delivered by minicells include, but are not limited to, RNAinterference molecule(s), or ribozyme(s), double stranded therapeuticRNA (e.g., dsRNA or siRNA), single stranded therapeutic RNA (e.g.,shRNA), aptamers, ribozymes, eukaryotic expression plasmids encoding fortherapeutic polypeptide(s) and/or therapeutic nucleic acids, and anycombination of the preceding. Recombinant expression of therapeuticnucleic acid(s) can be the result of expression from any of the variousepisomal recombinant prokaryotic expression vectors known in the artincluding, but not limited to, plasmids, cosmids, phagemids, andbacterial artificial chromosomes (BACs), and any combination of thepreceding. Recombinant expression can also be achieved by achromosomally located prokaryotic expression cassette present in one ormore copies of the minicell-producing parent cell chromosome. In caseswhere the therapeutic nucleic acid molecule(s) to be delivered exerttheir therapeutic effects through a “gene silencing” mechanism ofaction, the therapeutic nucleic acids are specific for one or moredifferent eukaryotic mRNA transcripts. The therapeutic nucleic acids canbe delivered by the same minicell such that one or more genes aresilenced by a single delivery event. Targeted minicells are also used todeliver any of these therapeutic nucleic acids in combination. Inaddition, targeted minicells are used to deliver one or more smallmolecule drugs in concert with one or more therapeutic nucleic acids.

Effective delivery of therapeutic nucleic acids by way of receptormediated endocytosis can be limited if the nucleic acid(s) delivered areexposed to the nuclease and protease rich environment of the endosomalcompartment for too long prior to being released to the cytosol of thetargeted eukaryotic cell. Thus, the skilled artisans will appreciatethat enhanced endosomal escape of therapeutic nucleic acids delivered bythis route may be desirable. However, this is not always necessary andis included per the discretion of the skilled artisan and can also beemployed in the delivery of other targeted minicell-borne payloadsincluding, but not limited to, small molecules, peptides, proteins, andradionuclides and any combination of the preceding. Intracellularpathogens are faced with the same problem and as a result have evolvedsophisticated mechanisms to either modulate the environment of theendosome to make it hospitable or to escape the endosome completely. Inthe case of the latter, this is typically mediated by a proteincomponent or protein complex made by the invading organism. Thelisteriolysin O (LLO; SEQ ID N0:29) protein of the intracellularGram-positive pathogenic bacterium Listeria monocytogenes is of interestand is incorporated into those embodiments of the present applicationthat include but are not limited to a therapeutic nucleic acid payloadcomponent. When expressed in an Fc-binding minicell producing bacterialstrain that also expresses therapeutic nucleic acid(s), LLO can beco-encapsulated with the therapeutic nucleic acid(s) by the Fc-bindingminicells which are subsequently made targeting-competent by addition ofan antibody or Fc-containing fusion/conjugate molecule to the surface ofthe Fc-binding minicells. Upon targeting of the minicell(s), receptormediated endocytosis carries the minicell into the endosome. The harshenvironment of the endosome begins to degrade the engulfed minicell,co-releasing the therapeutic nucleic acid payload along with LLO. Thereleased LLO component then facilitates release of the therapeuticnucleic acid from the endosome into the cytosol where the nucleic acidcan exert its biological effect(s).

In cases where the therapeutic nucleic acid molecule(s) is pre-formed bythe parental cell by way of recombinant expression from a prokaryoticexpression cassette (either chromosomal or episomal in location) and isthen packaged inside of the minicells as double stranded RNAs (e.g.,siRNA) or single stranded RNAs capable of folding back on themselves toform hairpin structures (e.g., shRNAs), the half-life of the therapeuticRNA(s) within the minicell is increased by use of Fc-binding minicellproducing bacterial strains that harbor a deletion or othernon-functional mutation in RNase genes (e.g., prokaryotic RNase III)responsible for the degradation of intracellular double stranded and/orhairpin RNA molecules. In the absence of the RNase, the therapeutic RNAmolecules accumulate to a higher level, increasing the potency oftargeted minicells delivering the therapeutic nucleic acid molecules. Inthe case of Escherichia coli minicell producing strains, mutation ordeletions are introduced into the mc gene, which encodes for the onlyknown somatic RNaseIII in this species.

Recombinantly expressed/produced therapeutic polypeptides to bedelivered by targeted minicells include but are not limited to proteintoxins, cholesterol-dependent cytolysins, functional enzymes, activatedcaspases, pro-caspases, cytokines, chemokines, cell-penetratingpeptides, and any combination of the preceding examples. Recombinantexpression of a therapeutic polypeptide(s) can be the result ofexpression from any of the various episomal recombinant prokaryoticexpression vectors known in the art including but not limited toplasmids, cosmids, phagemids, and bacterial artificial chromosomes(BACs), and any combination of the preceding examples. In similarfashion, recombinant expression can be achieved by a chromosomallylocated prokaryotic expression cassette present in one or more copies ofthe minicell-producing parent cell chromosome. The delivery of proteintoxins using the targeted minicells of the present application is anadvantageous approach in applications where selective elimination ofcells in vivo is desirable. Protein toxins which can facilitateendosomal delivery of payloads and/or function as toxic payloadsinclude, but are not limited to, fragments A/B of diphtheria toxin,fragment A of diphtheria toxin, anthrax toxins LF and EF, adenylatecyclase toxin, gelonin, botulinolysin B, botulinolysin E3, botulinolysinC, botulinum toxin, cholera toxin, clostridium toxins A, B and alpha,ricin, shiga A toxin, shiga-like A toxin, cholera A toxin, pertussis S1toxin, perfringolysin O, Pseudomonas exotoxin A, E. coli heat labiletoxin (LTB), melittin, pH stable variants of listeriolysin O(pH-independent; amino acid substitution L461T), thermostable variantsof listeriolysin O (amino acid substitutions E247M, D320K), pH andthermostable variants of listeriolysin O (amino acid substitutionsE247M, D320K, and L461T), streptolysin O, streptolysin O c, streptolysinO e, sphaericolysin, anthrolysin O, cereolysin, thuringiensilysin O,weihenstephanensilysin, alveolysin, brevilysin, butyriculysin,tetanolysin O, novyilysin, lectinolysin, pneumolysin, mitilysin,pseudopneumolysin, suilysin, intermedilysin, ivanolysin, seeligeriolysinO, vaginolysin, and pyolysin. Therapeutic polypeptides can be localizedto different sub-cellular compartments of the minicell at the discretionof the skilled artisans. When targeted minicells disclosed herein arederived from a Gram-negative parental minicell-producing strain,recombinantly expressed therapeutic polypeptides produced therefrom canbe localized to the cytosol, the inner leaflet of the inner membrane,the outer leaflet of the inner membrane, the periplasm, the innerleaflet of the outer membrane, the outer membrane of minicells, and anycombination of the proceeding. When targeted minicells disclosed hereinare derived from a Gram-positive parental minicell-producing strain,recombinantly expressed therapeutic polypeptides produced therefrom canbe localized to the cytosol, the cell wall, the inner leaflet of themembrane, the membrane of minicells, and any combination of theproceeding.

Effective delivery of therapeutic polypeptides by way of receptormediated endocytosis can be limited if the polypeptide(s) delivered areexposed to the protease rich environment of the endosomal compartmentfor too long prior to being released to the cytosol of the targetedeukaryotic cell. Indeed, most therapeutic polypeptides have no intrinsicability to escape the endosomal compartment, the exception being thecholesterol dependent cytolysins/toxins (e.g. LLO, perfringolysin O(PFO), and streptolysin O (SLO)) as well as fragment A/B of diphtheriatoxin (escape mediated by fragment B), ricin, and Pseudomonas exotoxinA. Those protein toxins that do contain intrinsic endosomal escapeproperties do not necessarily require the co-presence of a separateendosomal disruption component in the targeted minicell to be effectiveand the decision to include an endosomal disrupting agent is at thediscretion of the skilled artisans. Other protein toxins, such asgelonin and fragment A of the diphtheria toxin, have no intrinsicability to escape the endosomal compartment. Thus, the skilled artisanswould recognize that enhanced endosomal escape of many differenttherapeutic polypeptides delivered by the endosomal route is desirable.As described above, the listeriolysin O (LLO) protein of theintracellular Gram-positive pathogenic bacterium Listeria monocytogenesis of interest and is incorporated into those embodiments of the presentapplication that include but are not limited to a therapeuticpolypeptide payload component or other therapeutic payload requiringendosomal escape to confer best activity. In some embodiments, fulllength LLO (containing the signal secretion sequence) is used as theendosomal disruption agent. In some embodiments, the signal sequence ofLLO (making cLLO; SEQ ID NO:30) is removed at the genetic level usingrecombinant techniques known in the art and cLLO is used as theendosomal disruption agent. In some embodiments, thermostable and/orpH-independent versions of LLO (harboring mutations E247M, D320K and/orL461T, sLLOpH; SEQ ID NOs: 31 and 32, respectively) are employed. Whenexpressed in an Fc-binding minicell producing bacterial strain that alsoexpresses therapeutic polypeptide(s), LLO (or any of the LLO variants orother endosomal escape facilitators) can be co-encapsulated with thetherapeutic polypeptide(s) within the Fc-binding, minicells which aresubsequently made targeting-competent by addition of an antibody and/oran Fc-containing fusion/conjugate targeting molecule to the surface ofthe Fc-binding minicells. Upon targeting of the minicell(s), receptormediated endocytosis carries the minicell into the endosome. The harshenvironment of the endosome begins to degrade the engulfed minicell,co-releasing the therapeutic payload along with the endosomal disruptionagent (e.g., LLO, any of its variants, or other endosomal disruptingagent). The released endosomal disruption agent component thenfacilitates release of the therapeutic payload from the endosome intothe cytosol where the payload can exert its biological effect(s). Inaddition to LLO, preferred endosomal disruption agents include othercytolysins, such as PFO and SLO and derivatives thereof, andphospholipases, such as PI-PLC or PC-PLC.

In cases where the therapeutic polypeptide(s) is pre-formed by theparental cell by way of recombinant expression from a prokaryoticexpression cassette (either chromosomal or episomal in location) and isthen packaged inside of the minicells as the therapeutic payload, thehalf-life of the therapeutic polypeptide(s) within the minicell isincreased by use of Fc-binding minicell producing bacterial strains thatharbor a deletion or other non-functional mutation in protease genes(e.g., the Ion protease of E. coli) responsible for proteolysis. In theabsence of the protease(s), the therapeutic polypeptide(s) moleculeaccumulates to a higher level, increasing the potency of targetedminicells delivering the therapeutic polypeptide molecules. In the caseof Escherichia coli minicell producing strains, mutation or deletionscan be introduced into one or more of the lon, tonB, abgA, ampA, ampM,pepP, clpP, dcp, ddpX/vanX, elaD, frvX, gcp/b3064, hslV, hchA/b1967,hyaD, hybD, hycH, hycl, iadA, ldcA, ycbZ, pepD, pepE, pepQ, pepT, pmbA,pqqL, prlC, ptrB, sgcX, sprT, tldD, ycaL, yeaZ, yegQ, ygeY, yggG, yhbO,yibG, ydpF, degS, ftsH/hflB, glpG, hofD/hopD, lepB, lspA, pppA, sohB,spa, yaeL, yfbL, dacA, dacB, dacC, degP/htrA, degQ, iap, mepA,nlpC,pbpG, tsp, ptrA, teas, umuD, ydcP, ydgD, ydhO, yebA, yhbU, yhjJ,and nlpD genes.

In addition to being used as targeted small molecule drug andtherapeutic nucleic acid vehicles, the minicells disclosed herein canalso be used as targeted minicell vaccines. As described in more detailbelow, protein antigen and/or DNA vaccine loaded minicells are targeteddirectly to antigen presenting cells of the immune system by utilizingantibodies or Fc-containing fusion/conjugate molecules that are specificfor eukaryotic cell surface markers expressed by specific antigenpresenting cells. In some embodiments, it can be also desirable but notnecessary to include LLO or one of its variants (described above) tofacilitate transfer of antigen or DNA vaccine to the eukaryotic cellcytosol to promote MHC class-I loading, which stimulates cellularimmunity. It can also be desirable to promote MHC class-II loading tostimulate humoral (antibody mediated) immunity by keeping antigensinside the endosomal compartments where the large majority of MHC classII binding occurs. This can be accomplished by eliminating or decreasingthe LLO component of the targeted minicell vaccine. In addition,targeted vaccine minicells are further engineered to either express orbe loaded with exogenous adjuvant as deemed appropriate by the skilledartisan. Adjuvants can be general adjuvants (such as Keyhole limpethemocyanin or complete Freud's adjuvant) or can be targeted molecularadjuvants. Targeted molecular adjuvants include those that areantagonists or agonists of Toll-Like Receptors as well as other cellularconstituents that have immunomodulatory properties. Targeted vaccinesprovide recipient immunity to infectious disease agents including butnot limited to those infectious disease agents of bacterial, viral, andparasitic origin(s). Targeted vaccines also provided recipient immunityto tumors and other aberrant disease(s) of autologous nature.

In addition to being utilized as targeted delivery vehicles in vivo andin vitro, the Fc-binding minicells disclosed herein are also utilized asanalyte detection reagents for diagnostic assays including but notlimited to Lateral Flow Immunoassays (LFIAs). In some embodiments, theanalyte-detecting Fc-binding minicells can be comprised of (i)Fc-binding minicells, (ii) an analyte-specific antibody or otheranalyte-specific Fc-containing fusion/conjugate molecule bound to theFc-containing minicells, and (iii) a detection reagent including, butnot limited to, a small molecule flourophore, a fluorescent protein, anenzyme, a magnetic particle, and colloidal gold wherein the detectionreagent is encapsulated, displayed, or otherwise associated with theminicells. In a related permutation, Fc-binding minicells are used as anegative readout detection reagent for use in a competitive LFIA. Insome embodiments, negative readout Fc-binding minicells are comprised of(i) Fc-binding minicells, (ii) an Fc/analyte fusion/conjugate bound tothe Fc-containing minicells, and (iii) a detection reagent including butnot limited to a small molecule flourophore, a fluorescent protein, anenzyme, a magnetic particle, and colloidal gold wherein the detectionreagent is encapsulated, displayed, or otherwise associated with theminicells. Minicells can be used as detection reagents in kits used toanalyze clinical, veterinary, environmental, solid and liquidfoodstuffs, pharmaceutical products, and drinking water for the presenceor absence of a given relevant analyte in solution. Lateral FlowImmunoassays are constructed whereby they contain (i) product backing,(ii) a sample pad, (iii) a particle conjugate pad, (iv) a porousmembrane (e.g. nitrocellulose), (v) a test line, (vi) a control line,and (vii) a wick material. LFIAs are used as rapid point-of-carediagnostics as well as for in-home use (e.g., pregnancy tests), andvarious field tests (e.g. determining toxin levels in drinking water orsoil). LFIA detection reagents are currently limited to colloidal goldconjugates (10 nm), latex beads (colored or fluorescent; varying sizes),and paramagnetic latex covered beads (colored or fluorescent; varyingsizes). Each has its limitations and the need for new and improveddetection reagents is a key hurdle in the field at present. Colloidalgold conjugates are limited by their sensitivity and cost, latex beadsby their lack of sensitivity, and paramagnetic latex beads by theircost. Thus, there is a need for a cost-effective, highly sensitive classof particle-based detection reagents in the diagnostics field that willenable more quantitative and reliable assays, and/or a reduction inmanufacturing cost. Because minicells can be loaded with a wide varietyof different detection modalities, including functional enzymes that canamplify detection signals and increase sensitivity (not an option withcurrently available detection particles), they offer significantadvantages over currently utilized detection systems.

In order for targeted minicells to be used as therapeutic and diagnosticagents in humans, minicells should contain few or no contaminants, suchas viable parental bacterial cells. Levels of viable contaminating cellsand other contaminants must be low enough not to cause adverse sideeffects in patients or to interfere with minicell activity. Theinducible expression of a homing endonuclease gene, referred to as agenetic suicide mechanism, is a preferred mechanism by which toeliminate live contaminating parental cells, especially when used incombination with conventional filtration methods. Because minicells arederived from some bacteria that are pathogenic or opportunisticallypathogenic, it is important that any contaminating parental cells befunctionally eliminated from a given population before systemic, andparticularly intravenous, administration. Consequently, the desiredminicell formulation would be one in which the residual live parentalcell count would be as low as possible so as not cause adverse sideeffects or interfere with intended minicell activity. To minimize safetyconcerns, the minicells disclosed herein are derived fromminicell-producing strains that comprise safety features, for example,one or more of the three safety features disclosed below. In someembodiments, the minicell-producing strains comprise at least thesethree synergistic safety features. The first is a genetic suicidemechanism that kills residual live parental cells without lysing them(and expelling free lipopolysaccharide) after the minicell formationstep has been completed. The present application incorporates the use ofa regulated genetic suicide mechanism that upon exposure to theappropriate inducer, introduces irreparable damage to the chromosomes ofminicell-producing parental cells as described in U.S. PatentPublication No. 20100112670, which is hereby incorporated by referencein its entirety. The suicide mechanism operates to introduce irreparabledouble-stranded breaks to the chromosome of the parental cells and isused as an adjunct to conventional separation techniques to improveminicell purification. The second safety feature is a definedauxotrophy, preferably but not necessarily in the diaminopimelic acid(DAP) biosynthesis pathway, and most preferably in the dapA gene of anE. coli minicell-producing strain. Minicell-producing strains of E. colithat exhibit DAP auxotrophy (dapA-) cannot survive outside of thelaboratory without supplementation of DAP. Further, DAP is not found inmammals, including humans, and as such any minicell-producing parentalcells that happen to escape the genetic suicide mechanism will not beable to survive in the environment or in vivo. Many variations on thistheme exist for different Gram-negative and Gram-positive bacteria. Forexample in Salmonella, spp., auxotrophies in the aromatic amino acidbiosynthesis pathways (the aro genes) produce in effect, the sameresult. In the case of Shigella spp. auxotrophies in the guaninebiosynthesis pathway will produce, in effect, the same result. The thirdsafety feature is optional and entails a deletion of the lpxM gene in E.coli minicell-producing strains. Deletion of the lpxM gene can result inthe production of de-toxified lipopolysaccharide (LPS) molecules. ThelpxM gene (also referred to as the msbB gene) functions to add aterminal myristolic acid group to the lipid A portion of the LPSmolecule and removal of this group (by way of elimination of the lpxMgene) results in marked detoxification of LPS. Specifically,detoxification is characterized by a decrease in the production ofpro-inflammatory cytokines in response to exposure to LPS. This deletioncan be introduced into any functionally equivalent gene of anyGram-negative or Gram-positive minicell-producing strain to achieve thesame effect. The enhanced safety profile can reduce the risk ofinfection and potential for developing sepsis, decrease the possibilityof genetic reversion through recombination events with other bacteria,and minimize the risk of insertion events in the host. From a regulatoryand manufacturing perspective, it is also preferred that antibioticresistance markers be eliminated from the bacterial chromosome of theminicell-producing parental cell strain. The use of most antibioticresistance gene markers in minicell-producing strains of bacteria isundesirable in order to comply with regulatory requirements imposed bythe U.S. Food and Drug Administration (FDA) for use in humans. The FDAwill only tolerate the use of the kanamycin resistance gene marker forselection purposes for bacteria or bacterial production strains whereinthe final product is intended for use in humans.

As described herein, Fc-binding eubacterial minicells are capable ofbeing made targeting competent and delivering several classes ofbioactive payload in concert or singular wherein the final preparationof minicells is comprised of detoxified LPS and is sufficiently devoidof any viable contaminating parent cells by virtue of the combinedeffects of a novel, inducible genetic suicide mechanism used inconjunction with conventional separation techniques.

As described herein, bacterial minicells can be used as targeted in vivotherapeutic delivery, diagnostic, theranostic, and imaging agents. Insome embodiments, bacterial minicells are designed to incorporate abioactive payload, and by way of a novel mechanism, readily displayantibodies and/or other Fc-containing molecular targetingfusions/conjugates on their surfaces that specifically target theminicell to cell types involved in the initiation, promotion, support,and maintenance of disease in an animal. Some embodiments provideminicells that express and display the Fc binding region of Protein G onthe minicell surface wherein the minicell further comprises an antibodyand/or Fc-containing fusion/conjugate targeting molecule specific for aeukaryotic cell surface receptor bound by its Fc region to the Fcbinding portion of Protein G on the minicell surface wherein theantibody and/or Fc-containing fusion/conjugate targeting molecule-coatedminicell further comprises a bioactive payload(s) including but notlimited to a small molecule drug, a therapeutic nucleic acid, aradionuclide, an imaging agent, a protein, and any combination of thepreceding bioactive payloads. Some embodiments provide minicells thatexpress and display the Fc binding region of Protein A on the minicellsurface wherein the minicell further comprises an antibody and/orFc-containing fusion/conjugate targeting molecule specific for aeukaryotic cell surface receptor, wherein the antibody or Fc-containingfusion/conjugate targeting molecule is bound by its Fc region to the Fcbinding portion of Protein A on the minicell surface wherein theantibody and/or Fc-containing fusion/conjugate targeting molecule-coatedminicell further comprises a bioactive payload(s) including but notlimited to a small molecule drug, a therapeutic nucleic acid, aradionuclide, an imaging agent, a protein, and any combination of thepreceding bioactive payloads.

Some embodiments provide minicells that express and display the Fcbinding region of Protein G on the minicell surface wherein the minicellfurther comprises an antibody and/or Fc-containing fusion/conjugatetargeting molecule specific for a eukaryotic cell surface receptor,wherein the antibody or Fc-containing fusion/conjugate targetingmolecule is bound by its Fc region to the Fc binding portion of ProteinG on the minicell surface wherein the antibody and/or Fc-containingfusion/conjugate targeting molecule-coated minicell further comprises abioactive payload(s) including but not limited to a small molecule drug,a therapeutic nucleic acid, a radionuclide, an imaging agent, a protein,and any combination of the preceding bioactive payloads.

As described herein, bacterial minicells can be used as diagnostic testdetection reagents. Such reagents can be utilized as the detectionreagent in a wide variety of point-of-care/point-of-need diagnosticproduct types including but not limited to Lateral Flow Immunoassays. Insome embodiments, bacterial minicells can be designed to incorporate adetection reagent and by way of a novel approach, readily displayantibodies and/or Fc-containing fusion/conjugate targeting molecules ontheir surfaces that confer specificity of the minicell detection reagentfor a particular analyte or series of analytes to be tested for. Someembodiments provide minicells that express and display the Fc bindingregion of Protein A on the minicell surface wherein the minicell furthercomprises an antibody specific for a eukaryotic cell surface receptor,wherein the antibody or Fc-containing fusion/conjugate targetingmolecule is bound by its Fc region to the Fc binding portion of ProteinA on the minicell surface wherein the antibody and/or Fc-containingfusion/conjugate targeting molecule coated minicell further comprises adetectable reagent(s) including but not limited to a small moleculeflourophore, a magnetic particle(s), a colloidal gold particle(s), anactive enzyme, a fluorescent protein, and any combination of thepreceding detection reagents.

As described herein, bacterial minicells can be used as diagnostic testdetection reagents. Such reagents can be utilized as the detectionreagent in a wide variety of point-of-care/point-of-need diagnosticproduct types including but not limited to Lateral Flow Immunoassays. Insome embodiments, bacterial minicells are designed to incorporate adetection reagent and by way of a novel approach, readily displayantibodies and/or Fc-containing fusion/conjugate targeting molecules ontheir surfaces that confer specificity of the minicell detection reagentfor a particular analyte or series of analytes to be tested for. Someembodiments provide minicells that express and display the Fc bindingregion of Protein G on the minicell surface wherein the minicell furthercomprises an antibody specific for a eukaryotic cell surface receptor,wherein the antibody or Fc-containing fusion/conjugate targetingmolecule is bound by its Fc region to the Fc binding portion of ProteinG on the minicell surface wherein the antibody and/or Fc-containingfusion/conjugate targeting molecule coated minicell further comprises adetectable reagent(s) including but not limited to a small moleculeflourophore, a magnetic particle(s), a colloidal gold particle(s), anactive enzyme, a fluorescent protein, and any combination of thepreceding detection reagents.

In some preferred embodiments, bacterial minicells are used as targetedbioactive molecule delivery vehicles in vivo. In some embodiments,targeted therapeutic minicells comprise a bioactive (synonymous withbiologically active) payload that has a negative and/or therapeuticeffect on a cell that is involved in disease or another aberrant processin an animal. In some embodiments, the bioactive payload includes but isnot limited to small molecule drugs, bioactive nucleic acids, bioactiveproteins, bioactive radionuclides, imaging agents, and bioactivelipopolysaccharides, and any combination of the proceeding to produce a“biological effect” (synonymous with biological response) thatnegatively impacts diseased cells, tissues, or organs or positivelyeffects the production of signals that indirectly mitigate diseasedcells, tissues, or organs in an animal. In some embodiments, targetedminicells have biological effects that negatively impact diseaseincluding but not limited to an effect that kills cells responsible forthe initiation, promotion, or maintenance of the disease; an effect thatpositively impacts the production of signals that mitigate disease in ananimal; an effect that negatively impacts a biological processresponsible for the activation of disease in an animal; an effect thatelicits an innate immune response in an animal that negatively effectsdisease, and an effect that elicits an adaptive (humoral and/orcellular) response that negatively impacts disease in an animal, totreat or prevent a disease in the animal. In some embodiments, targetedminicells have biological effects that synergistically negatively impactdisease in an animal by exerting a combination of any of the biologicaleffects listed above. In some embodiments, the targeting moiety is anantibody, Fc-containing antibody derivative, and/or Fc-containingfusion/conjugate targeting molecule that is bound to the Fc-bindingregion of either Protein A or Protein G that is expressed and displayedon the surface of the minicells in the context of a contiguous fusionprotein that comprises (i) an outer membrane export (secretion) sequence(ii) an outer membrane protein or membrane anchoring portion thereof,and (iii) the Fc binding portion(s) of Protein A or Protein G on theminicell surface. Minicells displaying the Fc binding region(s) ofProtein A or Protein G can bind full length antibodies, Fc-containingantibody derivatives, and/or Fc-containing fusion/conjugate targetingmolecules through interaction with the Fc region of the molecules. Insome embodiments, the binding portion(s) of Protein A or Protein G ispart of a fusion protein designed to be expressed and displayed on thesurfaces of minicells. In some embodiments, the binding portion(s) ofProtein A or Protein G is a fusion protein with the Neisseria gonnorehaeIgAP autotransporter protein. In some embodiments, the bindingportion(s) of Protein A or Protein G is a fusion with a putative orpredicted outer membrane protein found in gram negative bacteria asdescribed in more detail herein. In some embodiments, the bindingportion(s) of Protein A or Protein G is a fusion with the Lpp-OmpAdisplay system which is described in U.S. Pat. No. 5,348,867 and herebyincorporated by reference in its entirety (SEQ ID NO.22 and SEQ IDNO:23, respectively). The antibody, Fc-containing antibody derivative,and/or Fc-containing fusion/conjugate targeting molecule on the surfaceof minicells can preferentially recognize but is not limited torecognizing cell-specific surface antigens including α3β1 integrin, α4β1integrin, α5β1 integrin, α_(v)β3 integrin, α_(v)β1 integrin, β1integrin, 5T4, CAIX, CD4, CD13, CD19, CD20, CD22, CD25, CD30, CD31,CD33, CD34, CD40, CD44v6, CD45, CD51, CD52, CD54, CD56, CD64, CD70,CD74, CD79, CD105, CD117, CD123, CD133, CD138, CD144, CD146, CD152,CD174, CD205, CD227, CD326, CD340, Cripto, ED-B, GD2, TMEFF2, VEGFR1,VEGFR2, FGFR, PDGFR, ANGPT1, TIE1, TIE2, NRP1, TEK (CD202B), TGFβR,Death Receptor 5 (Trail-R2), DLL4, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5,EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5,EPHB6, FAP, GPNMB, ICAMs, VCAMs, PSMA, HER-2/neu, IL-13R alpha 2, MUC-1,MUC16, EGFR1 (HER-1), EGFR2 (HER-2/neu), EGFR3 (HER-3), IGF-1R, IGF-2R,c-Met (HGFR), Mesothelin, PDGFR, EDGR, TAG-72, transferrin receptor,EpCAM, CTLA-4, PSMA, tenascin C, alpha-fetoprotein, vimentin, C242antigen, TRAIL-R1, TRAIL-R2, CA-125, GPNMB, CA-IX, GD3 ganglioside,RANKL, BAFF, IL-6R, TAG-72, HAMA, and CD166. In some embodiments, thetargeting moiety is selected, in part, because the binding of theminicell-surface displayed antibody targeting moiety, Fc-containingantibody derivatives, and/or Fc-containing fusion/conjugate targetingmolecules specific for the antigen induce internalization of thetargeted minicell, facilitating intracellular payload delivery.Previously described target-specific antibodies that are used as thetargeting component, in some embodiments, include but are not limited tomAb 3F8, mAb CSL362, mAb CSL360, mAb J591, Abagovomab, Abciximab,Adalimumab, Afelimomab, Afutuzumab, Alacizumab, ALD518, Alemtuzumab,Altumomab, Anatumomab, Anrukinzumab, Apolizumab, Arcitumomab,Aselizumab, Atlizumab, Atorolimumab, Bapineuzmab, Basiliximab,Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab,Besilesomab, Bevacizumab, Biciromab, Bivatuzumab, Blinatumomab,Brentuximab, Briakinumab, Canakinumab, Cantuzumab, Capromab,Catumaxomab, CC49, Cedelizumab, Certolizumab, Cetuximab, mAb528,Citatuzumab, Cixutumumab, Clenoliximab, Clivatuzumab, Conatumumab,CR6261, Dacetuzumab, Daclizumab, Daratumumab, Denosumab, Detumomab,Dorlimomab, Dorlixizumab, Ecromeximab, Eculizumab, Edobacomab,Edrecolomab, Efalizumab, Efungumab, Elotuzumab, Elsilimomab, Enlimomab,Epitumomab, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab,Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Felvizumab,Fezakinumab, Figitumumab, Fontolizumab, Foravirumab, Fresolimumab,Galiximab, Gantenerumab, Gavilimomab, Gemtuzumab, Girentuximab,Glembatumumab, Golimumab, Gomiliximab, Ibalizumab, Irbitumomab,Igovomab, Imciromab, Infliximab, Intetumumab, Inolimomab, Inotuzumab,Ipilimumab, Iratumumab, J591, Keliximab, Labetuzumab, Lebrikizumab,Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Lintuzumab,Lorvotuzumab, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab,Matuzumab, Mepolizomab, Metelimumab, Milatuzumab, Minretumomab,Mitumomab, Morolimumab, Motavizumab, Muromonab, Nacolomab, Naptumomab,Natalizumab, Nebacumab, Necitutumab, Nerelimomab, Nimotuzumab,Nofetumomab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab,Omalizumab, Oportuzumab, Oregovomab, Otelixizumab, Pagibaximab,Palivizumab, Panitumumab, Panobacumab, Pascolizumab, Pemtumomab,Pertuzumab, Pexelizumab, Pintumomab, Priliximab, Pritumumab, PRO140,Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab,Resilizumab, Rilotumumab, Rituximab, Robatumumab, Rontalizumab,Rovelizumab, Ruplizumab, Satumomab, Sevirumab, Sibrotuzumab,Sifalimumab, Siltuximab, Siplizumab, Solanezumab, Sonepcizumab,Sontuzumab, Stamulumab, Sulesomab, Tacatuzumab, Tadocizumab, Talizumab,Tanezumab, Taplitumomab, Tefibazumab, Telimomab, Tenatumomab,Teplizumab, TGN1412, Ticilimumab, Tigatuzumab, TNX-650, Tocilizumab,Toralizumab, Tositumomab, Trastuzumab, Tremelimumab, Tucotuzumab,Tuvirumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab,Veltuzumab, Vepalimomab, Visilizumab, Volociximab, Votumumab,Zalutumumab, Zanolimumab, Ziralimumab, Zolimomab, and any combination ofthe preceding.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted small molecule delivery vehicles in vivo and are used toprevent, inhibit, and/or limit disease progression in an animal. In someembodiments, targeted minicells are (i) derived from Fc-bindingminicells wherein the Fc-binding minicells display the Fc bindingportion of Protein G on their surfaces, (ii) are loaded with one or morespecies of small molecule drugs, (iii) further comprise surfacelocalized antibodies and/or Fc-containing fusion/conjugate moleculesthat are bound to the Fc binding portion of the surface displayed Fcbinding region of Protein G wherein the antibodies and/or Fc-containingfusion/conjugate molecules that recognize a eukaryotic cell-specificsurface antigen and are capable of stimulating receptor mediatedendocytosis upon binding of the targeted minicell to the eukaryoticcell-specific surface antigen, and (iv) further comprise apharmaceutically acceptable carrier for intravenous administration. Thespecies of small molecule drug(s) are selected from but not limited to(1) DNA damaging agents and agents that inhibit DNA synthesis such asanthracyclines (doxorubicin, daunorubicin, epirubicin), alkylatingagents (bendamustine, busulfan, carboplatin, carmustine, cisplatin,chlorambucil, cyclophosphamide, dacarbazine, hexamethylmelamine,ifosphamide, lomustine, mechlorethamine, melphalan, mitotane, mytomycin,pipobroman, procarbazine, streptozocin, thiotepa, andtriethylenemelamine), platinum derivatives (cisplatin, carboplatin, cisdiamminedichloroplatinum), telomerase and topoisomerase inhibitors(Camptosar), (2) microtubule and tubulin binding agents including butnot limited to taxanes and taxane derivatives (paclitaxel, docetaxel,BAY 59-8862), (3) anti-metabolites such as capecitabine,chlorodeoxyadenosine, cytarabine (and its activated form, ara-CMP),cytosine arabinoside, dacarbazine, floxuridine, fludarabine,5-fluorouracil, 5-DFUR, gemcitabine, hydroxyurea, 6-mercaptopurine,methotrexate, pentostatin, trimetrexate, and 6-thioguanine (4)anti-angiogenics (thalidomide, sunitinib, lenalidomide), vasculardisrupting agents (flavonoids/flavones, DMXAA, combretastatinderivatives such as CA4DP, ZD6126, AVE8062A, etc.), (5) endocrinetherapy such as aromatase inhibitors (4-hydroandrostendione, exemestane,aminoglutethimide, anastrozole, letrozole), (6) anti-estrogens(Tamoxifen, Toremifene, Raloxifene, Faslodex), steroids such asdexamethasone, (7) immuno-modulators such as Toll-like receptor agonistsor antagonists, (8) inhibitors to integrins, other adhesion proteins andmatrix metalloproteinases), (9) histone deacetylase inhibitors, (10)inhibitors of signal transduction such as inhibitors of tyrosine kinaseslike imatinib (Gleevec), (11) inhibitors of heat shock proteins, (12)retinoids such as all trans retinoic acid, (13) inhibitors of growthfactor receptors or the growth factors themselves, (14) anti-mitoticcompounds such as navelbine, vinblastine, vincristine, vindesine, andvinorelbine, (15) anti-inflammatories such as COX inhibitors and (16)cell cycle regulators such as check point regulators and telomeraseinhibitors, (17) transcription factor inhibitors, and apoptosisinducers, such as inhibitors of Bcl-2, Bcl-x and XIAP and anycombination of the preceding (1-17). The antibody and/or Fc-containingfusion/conjugate molecules on the surface of minicells canpreferentially recognize but is not limited to recognizing cell-specificsurface antigens including α3β1 integrin, α4β1 integrin, α5β1 integrin,α_(v)β3 integrin, α_(v)β1 integrin, β1 integrin, 5T4, CAIX, CD4, CD13,CD19, CD20, CD22, CD25, CD30, CD31, CD33, CD34, CD40, CD44v6, CD45,CD51, CD52, CD54, CD56, CD64, CD70, CD74, CD79, CD105, CD117, CD123,CD133, CD138, CD144, CD146, CD152, CD174, CD205, CD227, CD326, CD340,Cripto, ED-B, GD2, TMEFF2, VEGFR1, VEGFR2, FGFR, PDGFR, ANGPT1, TIE1,TIE2, NRP1, TEK (CD202B), TGFβR, Death Receptor 5 (Trail-R2), DLL4,EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10,EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, FAP, GPNMB, ICAMs, VCAMs,PSMA, HER-2/neu, IL-13R alpha 2, MUC-1, MUC16, EGFR1 (HER-1), EGFR2(HER-2/neu), EGFR3 (HER-3), IGF-1R, IGF-2R, c-Met (HGFR), Mesothelin,PDGFR, EDGR, TAG-72, transferrin receptor, EpCAM, CTLA-4, PSMA, tenascinC, alpha-fetoprotein, vimentin, C242 antigen, TRAIL-R1, TRAIL-R2,CA-125, GPNMB, CA-IX, GD3 ganglioside, RANKL, BAFF, IL-6R, TAG-72, HAMA,and CD166.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted small molecule delivery vehicles in vivo and are used toprevent, inhibit, and/or limit disease progression in an animal. In someembodiments, targeted minicells are (i) derived from Fc-bindingminicells wherein the Fc-binding minicells display the Fc bindingportion of Protein A on their surfaces, (ii) are loaded with one or morespecies of small molecule drugs, (iii) further comprise surfacelocalized antibodies and/or Fc-containing fusion/conjugate moleculesthat are bound to the Fc binding portion of the surface displayed Fcbinding region of Protein A wherein the antibodies and/or Fc-containingfusion/conjugate molecules recognize a eukaryotic cell-specific surfaceantigen and are capable of stimulating receptor mediated endocytosisupon binding of the targeted minicell to the eukaryotic cell-specificsurface antigen, and (iv) further comprise a pharmaceutically acceptablecarrier for intravenous administration. The species of small moleculedrug(s) are selected from but not limited to (1) DNA damaging agents andagents that inhibit DNA synthesis such as anthracyclines (doxorubicin,daunorubicin, epirubicin), alkylating agents (bendamustine, busulfan,carboplatin, carmustine, cisplatin, chlorambucil, cyclophosphamide,dacarbazine, hexamethylmelamine, ifosphamide, lomustine,mechlorethamine, melphalan, mitotane, mytomycin, pipobroman,procarbazine, streptozocin, thiotepa, and triethylenemelamine), platinumderivatives (cisplatin, carboplatin, cis diamminedichloroplatinum),telomerase and topoisomerase inhibitors (Camptosar), (2) microtubule andtubulin binding agents including but not limited to taxanes and taxanederivatives (paclitaxel, docetaxel, BAY 59-8862), (3) anti-metabolitessuch as capecitabine, chlorodeoxyadenosine, cytarabine (and itsactivated form, ara-CMP), cytosine arabinoside, dacarbazine,floxuridine, fludarabine, 5-fluorouracil, 5-DFUR, gemcitabine,hydroxyurea, 6-mercaptopurine, methotrexate, pentostatin, trimetrexate,and 6-thioguanine (4) anti-angiogenics (thalidomide, sunitinib,lenalidomide), vascular disrupting agents (flavonoids/flavones, DMXAA,combretastatin derivatives such as CA4DP, ZD6126, AVE8062A, etc.), (5)endocrine therapy such as aromatase inhibitors (4-hydroandrostendione,exemestane, aminoglutethimide, anastrozole, letrozole), (6)anti-estrogens (Tamoxifen, Toremifene, Raloxifene, Faslodex), steroidssuch as dexamethasone, (7) immuno-modulators such as Toll-like receptoragonists or antagonists, (8) inhibitors to integrins, other adhesionproteins and matrix metalloproteinases), (9) histone deacetylaseinhibitors, (10) inhibitors of signal transduction such as inhibitors oftyrosine kinases like imatinib (Gleevec), (11) inhibitors of heat shockproteins, (12) retinoids such as all trans retinoic acid, (13)inhibitors of growth factor receptors or the growth factors themselves,(14) anti-mitotic compounds such as navelbine, vinblastine, vincristine,vindesine, and vinorelbine, (15) anti-inflammatories such as COXinhibitors and (16) cell cycle regulators such as check point regulatorsand telomerase inhibitors, (17) transcription factor inhibitors, andapoptosis inducers, such as inhibitors of Bcl-2, Bcl-x and XIAP and anycombination of the preceding (1-17). The antibody and/or Fc-containingfusion/conjugate molecules on the surface of minicells canpreferentially recognize but are not limited to recognizingcell-specific surface antigens including α3β1 integrin, α4β1 integrin,α5β1 integrin, α_(v)β3 integrin, α_(v)β1 integrin, β1 integrin, 5T4,CAIX, CD4, CD13, CD19, CD20, CD22, CD25, CD30, CD31, CD33, CD34, CD40,CD44v6, CD45, CD51, CD52, CD54, CD56, CD64, CD70, CD74, CD79, CD105,CD117, CD123, CD133, CD138, CD144, CD146, CD152, CD174, CD205, CD227,CD326, CD340, Cripto, ED-B, GD2, TMEFF2, VEGFR1, VEGFR2, FGFR, PDGFR,ANGPT1, TIE1, TIE2, NRP1, TEK (CD202B), TGFβR, Death Receptor 5(Trail-R2), DLL4, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7,EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, FAP,GPNMB, ICAMs, VCAMs, PSMA, HER-2/neu, IL-13R alpha 2, MUC-1, MUC16,EGFR1 (HER-1), EGFR2 (HER-2/neu), EGFR3 (HER-3), IGF-1R, IGF-2R, c-Met(HGFR), Mesothelin, PDGFR, EDGR, TAG-72, transferrin receptor, EpCAM,CTLA-4, PSMA, tenascin C, alpha-fetoprotein, vimentin, C242 antigen,TRAIL-R1, TRAIL-R2, CA-125, GPNMB, CA-IX, GD3 ganglioside, RANKL, BAFF,IL-6R, TAG-72, HAMA, and CD166.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted therapeutic nucleic acid delivery vehicles in vivo and areused to prevent, inhibit, and/or limit disease progression in an animal.In some embodiments, targeted minicells are (i) derived from Fc-bindingminicells wherein the Fc-binding minicells display the Fc bindingportion of Protein G on their surfaces, (ii) are loaded with one or moretherapeutic nucleic acid molecules, (iii) further comprise surfacelocalized antibodies and/or Fc-containing fusion/conjugate moleculesthat are bound to the Fc binding portion of the surface displayed Fcbinding region of Protein G wherein the antibodies and/or Fc-containingfusion/conjugate molecules recognize a eukaryotic cell-specific surfaceantigen and are capable of stimulating receptor mediated endocytosisupon binding of the targeted minicell to the eukaryotic cell-specificsurface antigen, (iv) further comprise an endosomal disrupting agent,and (v) further comprise a pharmaceutically acceptable carrier forintravenous administration. In some embodiments, therapeutic nucleicacids that exert their effects by way of gene silencing (siRNA andshRNA, or a eukaryotic DNA expression plasmid encoding for the same)include but are not limited to comprising one or more contiguousnucleotide sequences having homology to wild-type gene sequences and/orto one or more contiguous sequences containing germ-line and somaticmutations known to be involved in disease, such as cancer. Thetherapeutic nucleic acid sequences are preferred to have twenty-two (22)nucleotides of homology to the target gene of interest. The therapeuticnucleic acid molecules can be directed against mRNA transcripts of genesincluding but not limited to Androgen Receptor (AR), ABCB1/MDR1/PGY1(P-glycoprotein; Pgp), CHK-1, HIF-1, Mcl-1, PDGFR, Tie-2, ABL1, ABL2,AKT2, ALK, BCL2, BCL3, BCL5, BCL6, BLC7A, BCL9, BCL10, BCL11A, BCL11B,Bcl-x, Bcr-Abl, BRAF, CCND1, CDK4, CHK-1, c-Met, c-myc, CTNNB1, DKC1,EGFR1, EGFR2, ERBB2, ERCC-1, EZH2, FES, FGFR1, FGFR2, FGFR3, FGFR-4,FLT1 (VEGFR1), FLT2, FLT3, FLT4, HER2, HER3, HRAS, IGFR, Interleukin 8(IL-8), JAK, JAK2, KDR/Flk-1 (VEGFR-2), KIT, KRAS2, MET, MRP, mTOR, MYC,MYCL1, MYCN, NRAS, p53, PARP1, PDGFB, PDGFRA, PDGFRB, PI3KCA, PPAR,Rad51, Rad52, Rad53, RalA, REL, RET, RRM1, RRM2, STAT3, survivin,telomerase, TEP1, TERC, TERT, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E,Wnt-1, and XIAP. The antibody and/or Fc-containing fusion/conjugatemolecules on the surface of minicells can preferentially recognize butare not limited to recognizing cell-specific surface antigens includingα3β1 integrin, α4β1 integrin, α5β1 integrin, α_(v)β3 integrin, α_(v)β1integrin, β1 integrin, 5T4, CAIX, CD4, CD13, CD19, CD20, CD22, CD25,CD30, CD31, CD33, CD34, CD40, CD44v6, CD45, CD51, CD52, CD54, CD56,CD64, CD70, CD74, CD79, CD105, CD117, CD123, CD133, CD138, CD144, CD146,CD152, CD174, CD205, CD227, CD326, CD340, Cripto, ED-B, GD2, TMEFF2,VEGFR1, VEGFR2, FGFR, PDGFR, ANGPT1, TIE1, TIE2, NRP1, TEK (CD202B),TGFβR, Death Receptor 5 (Trail-R2), DLL4, EPHA1, EPHA2, EPHA3, EPHA4,EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4,EPHB5, EPHB6, FAP, GPNMB, ICAMs, VCAMs, PSMA, HER-2/neu, IL-13R alpha 2,MUC-1, MUC16, EGFR1 (HER-1), EGFR2 (HER-2/neu), EGFR3 (HER-3), IGF-1R,IGF-2R, c-Met (HGFR), Mesothelin, PDGFR, EDGR, TAG-72, transferrinreceptor, EpCAM, CTLA-4, PSMA, tenascin C, alpha-fetoprotein, vimentin,C242 antigen, TRAIL-R1, TRAIL-R2, CA-125, GPNMB, CA-IX, GD3 ganglioside,RANKL, BAFF, IL-6R, TAG-72, HAMA, and CD166.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted therapeutic nucleic acid delivery vehicles in vivo and areused to prevent, inhibit, and/or limit disease progression in an animal.In some embodiments, targeted minicells are (i) derived from Fc-bindingminicells wherein the Fc-binding minicells display the Fc bindingportion of Protein A on their surfaces, (ii) are loaded with one or moretherapeutic nucleic acids, (iii) further comprise surface localizedantibodies and/or Fc-containing fusion/conjugate molecules that arebound to the Fc binding portion of the surface displayed Fc bindingregion of Protein A wherein the antibodies and/or Fc-containingfusion/conjugate molecules recognize a eukaryotic cell-specific surfaceantigen and are capable of stimulating receptor mediated endocytosisupon binding of the targeted minicell to the eukaryotic cell-specificsurface antigen, (iv) further comprise an endosomal disrupting agent,and (v) further comprise a pharmaceutically acceptable carrier forintravenous administration. In some embodiments, therapeutic nucleicacids that exert their effects by way of gene silencing (siRNA andshRNA, or a eukaryotic DNA expression plasmid encoding for the same)include but are not limited to comprising one or more contiguousnucleotide sequences having homology to wild-type gene sequences and/orto one or more contiguous sequences containing germ-line and somaticmutations known to be involved in disease, such as cancer. Thetherapeutic nucleic acid sequences are preferred to have twenty-two (22)nucleotides of homology to the target gene of interest. The therapeuticnucleic acid molecules may be directed against mRNA transcripts of genesincluding but not limited to Androgen Receptor (AR), ABCB1/MDR1/PGY1(P-glycoprotein; Pgp), CHK-1, HIF-1, Mcl-1, PDGFR, Tie-2, ABL1, ABL2,AKT2, ALK, BCL2, BCL3, BCL5, BCL6, BLC7A, BCL9, BCL10, BCL11A, BCL11B,Bcl-x, Bcr-Abl, BRAF, CCND1, CDK4, CHK-1, c-Met, c-myc, CTNNB1, DKC1,EGFR1, EGFR2, ERBB2, ERCC-1, EZH2, FES, FGFR1, FGFR2, FGFR3, FGFR-4,FLT1 (VEGFR1), FLT2, FLT3, FLT4, HER2, HER3, HRAS, IGFR, Interleukin 8(IL-8), JAK, JAK2, KDR/Flk-1 (VEGFR-2), KIT, KRAS2, MET, MRP, mTOR, MYC,MYCL1, MYCN, NRAS, p53, PARP1, PDGFB, PDGFRA, PDGFRB, PI3KCA, PPAR,Rad51, Rad52, Rad53, RalA, REL, RET, RRM1, RRM2, STAT3, survivin,telomerase, TEP1, TERC, TERT, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E,Wnt-1, and XIAP. The antibody and/or Fc-containing fusion/conjugatemolecules on the surface of minicells can preferentially recognize butare not limited to recognizing cell-specific surface antigens includingα3β1 integrin, α4β1 integrin, α5β1 integrin, α_(v)β3integrin, α_(v)β1integrin, β1 integrin, 5T4, CAIX, CD4, CD13, CD19, CD20, CD22, CD25,CD30, CD31, CD33, CD34, CD40, CD44v6, CD45, CD51, CD52, CD54, CD56,CD64, CD70, CD74, CD79, CD105, CD117, CD123, CD133, CD138, CD144, CD146,CD152, CD174, CD205, CD227, CD326, CD340, Cripto, ED-B, GD2, TMEFF2,VEGFR1, VEGFR2, FGFR, PDGFR, ANGPT1, TIE1, TIE2, NRP1, TEK (CD202B),TGFβR, Death Receptor 5 (Trail-R2), DLL4, EPHA1, EPHA2, EPHA3, EPHA4,EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4,EPHB5, EPHB6, FAP, GPNMB, ICAMs, VCAMs, PSMA, HER-2/neu, IL-13R alpha 2,MUC-1, MUC16, EGFR1 (HER-1), EGFR2 (HER-2/neu), EGFR3 (HER-3), IGF-1R,IGF-2R, c-Met (HGFR), Mesothelin, PDGFR, EDGR, TAG-72, transferrinreceptor, EpCAM, CTLA-4, PSMA, tenascin C, alpha-fetoprotein, vimentin,C242 antigen, TRAIL-R1, TRAIL-R2, CA-125, GPNMB, CA-IX, GD3 ganglioside,RANKL, BAFF, IL-6R, TAG-72, HAMA, and CD166.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted therapeutic polypeptide delivery vehicles in vivo and areused to prevent, inhibit, and/or limit disease progression in an animal.In some embodiments, targeted minicells are (i) derived from Fc-bindingminicells wherein the Fc-binding minicells display the Fc bindingportion of Protein A on their surfaces, (ii) are loaded with one or moretherapeutic polypeptides, (iii) further comprise surface localizedantibodies and/or Fc-containing fusion/conjugate molecules that arebound to the Fc binding portion of the surface displayed Fc bindingregion of Protein A wherein the antibodies and/or Fc-containingfusion/conjugate molecules recognize a eukaryotic cell-specific surfaceantigen and are capable of stimulating receptor mediated endocytosisupon binding of the targeted minicell to the eukaryotic cell-specificsurface antigen, (iv) further comprise an endosomal disrupting agent,and (v) further comprise a pharmaceutically acceptable carrier forintravenous administration. In some embodiments, therapeuticpolypeptides that exert their effects by way of cellular toxicity(protein toxins) include but are not limited to cholesterol dependentcytolysins, ADP-ribosylating toxins, plant toxins, bacterial toxins,viral toxins, pore forming toxins, and cell penetrating peptides. Thetherapeutic polypeptides can be selected from the group including butnot limited to gelonin, diphtheria toxin fragment A, diphtheria toxinfragment A/B, tetanus toxin, E. coli heat labile toxin (LTI and/orLTII), cholera toxin, C. perfringes iota toxin, Pseudomonas exotoxin A,shiga toxin, anthrax toxin, MTX (B. sphaericus mosquilicidal toxin),perfringolysin O, streptolysin, barley toxin, mellitin, anthrax toxinsLF and EF, adenylate cyclase toxin, botulinolysin B, botulinolysin E3,botulinolysin C, botulinum toxin A, cholera toxin, clostridium toxins A,B, and alpha, ricin, shiga A toxin, shiga-like A toxin, cholera A toxin,pertussis S1 toxin, E. coli heat labile toxin (LTB), pH stable variantsof listeriolysin O (pH-independent; amino acid substitution L461T),thermostable variants of listeriolysin O (amino acid substitutionsE247M, D320K), pH and thermostable variants of listeriolysin O (aminoacid substitutions E247M, D320K, and L461T), streptolysin O,streptolysin O c, streptolysin O e, sphaericolysin, anthrolysin O,cereolysin, thuringiensilysin O, weihenstephanensilysin, alveolysin,brevilysin, butyriculysin, tetanolysin O, novyilysin, lectinolysin,pneumolysin, mitilysin, pseudopneumolysin, suilysin, intermedilysin,ivanolysin, seeligeriolysin O, vaginolysin, and pyolysin The antibodyand/or Fc-containing fusion/conjugate molecules on the surface ofminicells can preferentially recognize but is not limited to recognizingcell-specific surface antigens including α3β1 integrin, α4β1 integrin,α5β1 integrin, α_(v)β3 integrin, α_(v)β1integrin, β1 integrin, 5T4,CAIX, CD4, CD13, CD19, CD20, CD22, CD25, CD30, CD31, CD33, CD34, CD40,CD44v6, CD45, CD51, CD52, CD54, CD56, CD64, CD70, CD74, CD79, CD105,CD117, CD123, CD133, CD138, CD144, CD146, CD152, CD174, CD205, CD227,CD326, CD340, Cripto, ED-B, GD2, TMEFF2, VEGFR1, VEGFR2, FGFR, PDGFR,ANGPT1, TIE1, TIE2, NRP1, TEK (CD202B), TGFβR, Death Receptor 5(Trail-R2), DLL4, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7,EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, FAP,GPNMB, ICAMs, VCAMs, PSMA, HER-2/neu, IL-13R alpha 2, MUC-1, MUC16,EGFR1 (HER-1), EGFR2 (HER-2/neu), EGFR3 (HER-3), IGF-1R, IGF-2R, c-Met(HGFR), Mesothelin, PDGFR, EDGR, TAG-72, transferrin receptor, EpCAM,CTLA-4, PSMA, tenascin C, alpha-fetoprotein, vimentin, C242 antigen,TRAIL-R1, TRAIL-R2, CA-125, GPNMB, CA-IX, GD3 ganglioside, RANKL, BAFF,IL-6R, TAG-72, HAMA, and CD166.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted therapeutic polypeptide delivery vehicles in vivo and areused to prevent, inhibit, and/or limit disease progression in an animal.In some embodiments, targeted minicells are (i) derived from Fc-bindingminicells wherein the Fc-binding minicells display the Fc bindingportion of Protein G on their surfaces, (ii) are loaded with one or moretherapeutic polypeptides, (iii) further comprise surface localizedantibodies and/or Fc-containing fusion/conjugate molecules that arebound to the Fc binding portion of the surface displayed Fc bindingregion of Protein G wherein the antibodies and/or Fc-containingfusion/conjugate molecules recognize a eukaryotic cell-specific surfaceantigen and are capable of stimulating receptor mediated endocytosisupon binding of the targeted minicell to the eukaryotic cell-specificsurface antigen, (iv) further comprise an endosomal disrupting agent,and (v) further comprise a pharmaceutically acceptable carrier forintravenous administration. In some embodiments, therapeuticpolypeptides that exert their effects by way of cellular toxicity(protein toxins) include but are not limited to cholesterol dependentcytolysins, ADP-ribosylating toxins, plant toxins, bacterial toxins,viral toxins, pore forming toxins, and cell penetrating peptides. Thetherapeutic polypeptides may be selected from the group including butnot limited to gelonin, diphtheria toxin fragment A, diphtheria toxinfragment A/B, tetanus toxin, E. coli heat labile toxin (LTI and/orLTII), cholera toxin, C. perfringes iota toxin, Pseudomonas exotoxin A,shiga toxin, anthrax toxin, MTX (B. sphaericus mosquilicidal toxin),perfringolysin O, streptolysin, barley toxin, mellitin, anthrax toxinsLF and EF, adenylate cyclase toxin, botulinolysin B, botulinolysin E3,botulinolysin C, botulinum toxin A, cholera toxin, clostridium toxins A,B, and alpha, ricin, shiga A toxin, shiga-like A toxin, cholera A toxin,pertussis S1 toxin, E. coli heat labile toxin (LTB), pH stable variantsof listeriolysin O (pH-independent; amino acid substitution L461T),thermostable variants of listeriolysin O (amino acid substitutionsE247M, D320K), pH and thermostable variants of listeriolysin O (aminoacid substitutions E247M, D320K, and L461T), streptolysin O,streptolysin O c, streptolysin O e, sphaericolysin, anthrolysin O,cereolysin, thuringiensilysin O, weihenstephanensilysin, alveolysin,brevilysin, butyriculysin, tetanolysin O, novyilysin, lectinolysin,pneumolysin, mitilysin, pseudopneumolysin, suilysin, intermedilysin,ivanolysin, seeligeriolysin O, vaginolysin, and pyolysin. The antibodyand/or Fc-containing fusion/conjugate molecule on the surface ofminicells can preferentially recognize but is not limited to recognizingcell-specific surface antigens including α3β1 integrin, α4β1 integrin,α5β1 integrin, α_(v)β3 integrin, α_(v)β1 integrin, β1 integrin, 5T4,CALX, CD4, CD13, CD19, CD20, CD22, CD25, CD30, CD31, CD33, CD34, CD40,CD44v6, CD45, CD51, CD52, CD54, CD56, CD64, CD70, CD74, CD79, CD105,CD117, CD123, CD133, CD138, CD144, CD146, CD152, CD174, CD205, CD227,CD326, CD340, Cripto, ED-B, GD2, TMEFF2, VEGFR1, VEGFR2, FGFR, PDGFR,ANGPT1, TIE1, TIE2, NRP1, TEK (CD202B), TGFβR, Death Receptor 5(Trail-R2), DLL4, EPHAL EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8,EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, FAP, GPNMB,ICAMs, VCAMs, PSMA, HER-2/neu, IL-13R alpha 2, MUC-1, MUC16, EGFR1(HER-1), EGFR2 (HER-2/neu), EGFR3 (HER-3), IGF-1R, IGF-2R, c-Met (HGFR),Mesothelin, PDGFR, EDGR, TAG-72, transferrin receptor, EpCAM, CTLA-4,PSMA, tenascin C, alpha-fetoprotein, vimentin, C242 antigen, TRAIL-R1,TRAIL-R2, CA-125, GPNMB, CA-IX, GD3 ganglioside, RANKL, BAFF, IL-6R,TAG-72, HAMA, and CD166.

In some preferred embodiments, targeted diagnostic minicells are used astargeted diagnostic imaging agents in vivo and are used to diagnose,detect, and/or monitor disease in an animal. In some embodiments,targeted minicells are (i) derived from Fc-binding minicells wherein theFc-binding minicells display the Fc binding portion of Protein G ontheir surfaces, (ii) are loaded with one or more molecular imagingagents, (iii) further comprise surface localized antibodies and/orFc-containing fusion/conjugate molecules that are bound to the Fcbinding portion of the surface displayed Fc binding region of Protein Gwherein the antibodies and/or Fc-containing fusion/conjugate moleculesrecognize a eukaryotic cell-specific surface antigen and are capable ofstimulating receptor mediated endocytosis upon binding of the targetedminicell to the eukaryotic cell-specific surface antigen, and (iv)further comprise a pharmaceutically acceptable carrier for intravenousadministration. The antibody and/or Fc-containing fusion/conjugatemolecule(s) on the surface of minicells can preferentially recognize butis not limited to recognizing cell-specific surface antigens includingα3β1 integrin, α4β1 integrin, α5β1 integrin, α_(v)β3integrin,α_(v)β1integrin, β1 integrin, 5T4, CAIX, CD4, CD13, CD19, CD20, CD22,CD25, CD30, CD31, CD33, CD34, CD40, CD44v6, CD45, CD51, CD52, CD54,CD56, CD64, CD70, CD74, CD79, CD105, CD117, CD123, CD133, CD138, CD144,CD146, CD152, CD174, CD205, CD227, CD326, CD340, Cripto, ED-B, GD2,TMEFF2, VEGFR1, VEGFR2, FGFR, PDGFR, ANGPT1, TIE1, TIE2, NRP1, TEK(CD202B), TGFIβR, Death Receptor 5 (Trail-R2), DLL4, EPHA1, EPHA2,EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2,EPHB3, EPHB4, EPHB5, EPHB6, FAP, GPNMB, ICAMs, VCAMs, PSMA, HER-2/neu,IL-13R alpha 2, MUC-1, MUC16, EGFR1 (HER-1), EGFR2 (HER-2/neu), EGFR3(HER-3), IGF-1R, IGF-2R, c-Met (HGFR), Mesothelin, PDGFR, EDGR, TAG-72,transferrin receptor, EpCAM, CTLA-4, PSMA, tenascin C,alpha-fetoprotein, vimentin, C242 antigen, TRAIL-R1, TRAIL-R2, CA-125,GPNMB, CA-IX, GD3 ganglioside, RANKL, BAFF, IL-6R, TAG-72, HAMA, andCD166.

In some preferred embodiments, targeted diagnostic minicells are used astargeted diagnostic imaging agents in vivo and are used to diagnose,detect, and/or monitor disease in an animal. In some embodiments,targeted minicells are (i) derived from Fc-binding minicells wherein theFc-binding minicells display the Fc binding portion of Protein G ontheir surfaces, (ii) are loaded with one or more molecular imagingagents, (iii) further comprise surface localized antibodies and/orFc-containing fusion/conjugate molecules that are bound to the Fcbinding portion of the surface displayed Fc binding region of Protein Gwherein the antibodies and/or Fc-containing fusion/conjugate moleculesrecognize a eukaryotic cell-specific surface antigen and do notstimulate receptor mediated endocytosis upon binding of the targetedminicell to the eukaryotic cell-specific surface antigen, and (iv)further comprise a pharmaceutically acceptable carrier for intravenousadministration. The antibody and/or Fc-containing fusion/conjugatemolecule(s) on the surface of minicells can preferentially recognize butis not limited to recognizing cell-specific surface antigens includingα3β1 integrin, α4β1 integrin, α5β1 integrin, α_(v)β3integrin,α_(v)β1integrin, β1 integrin, 5T4, CAIX, CD4, CD13, CD19, CD20, CD22,CD25, CD30, CD31, CD33, CD34, CD40, CD44v6, CD45, CD51, CD52, CD54,CD56, CD64, CD70, CD74, CD79, CD105, CD117, CD123, CD133, CD138, CD144,CD146, CD152, CD174, CD205, CD227, CD326, CD340, Cripto, ED-B, GD2,TMEFF2, VEGFR1, VEGFR2, FGFR, PDGFR, ANGPT1, TIE1, TIE2, NRP1, TEK(CD202B), TGFβR, Death Receptor 5 (Trail-R2), DLL4, EPHA1, EPHA2, EPHA3,EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3,EPHB4, EPHB5, EPHB6, FAP, GPNMB, ICAMs, VCAMs, PSMA, HER-2/neu, IL-13Ralpha 2, MUC-1, MUC16, EGFR1 (HER-1), EGFR2 (HER-2/neu), EGFR3 (HER-3),IGF-1R, IGF-2R, c-Met (HGFR), Mesothelin, PDGFR, EDGR, TAG-72,transferrin receptor, EpCAM, CTLA-4, PSMA, tenascin C,alpha-fetoprotein, vimentin, C242 antigen, TRAIL-R1, TRAIL-R2, CA-125,GPNMB, CA-IX, GD3 ganglioside, RANKL, BAFF, IL-6R, TAG-72, HAMA, andCD166. Non-limiting examples of the molecular imaging agents includeGadolinium, ⁶⁴Cu diacetyl-bis(N⁴-methylthiosemicarbazone),¹⁸F-flourodeoxyglucose, ¹⁸F-flouride, 3′-deoxy-3′-[¹⁸F]fluorothymidine,¹⁸F-fluoromisonidazole, gallium, technetium-99, thallium, barium,gastrografin, iodine contrasting agents, iron oxide, green fluorescentprotein, luciferase, beta-galactosidase, and any combination of thepreceding.

In some preferred embodiments, targeted diagnostic minicells are used astargeted diagnostic imaging agents in vivo and are used to diagnose,detect, and/or monitor disease in an animal. In some embodiments,targeted minicells are (i) derived from Fc-binding minicells wherein theFc-binding minicells display the Fc binding portion of Protein A ontheir surfaces, (ii) are loaded with one or more molecular imagingagents, (iii) further comprise surface localized antibodies and/orFc-containing fusion/conjugate molecules that are bound to the Fcbinding portion of the surface displayed Fc binding region of Protein Awherein the antibodies and/or Fc-containing fusion/conjugate moleculesrecognize a eukaryotic cell-specific surface antigen and are capable ofstimulating receptor mediated endocytosis upon binding of the targetedminicell to the eukaryotic cell-specific surface antigen, and (iv)further comprise a pharmaceutically acceptable carrier for intravenousadministration. The antibody and/or Fc-containing fusion/conjugatemolecule(s) on the surface of minicells can preferentially recognize butis not limited to recognizing cell-specific surface antigens includingα3β1 integrin, α4β1 integrin, α5β1 integrin, α_(v)β3integrin,α_(v)β1integrin, β1 integrin, 5T4, CAIX, CD4, CD13, CD19, CD20, CD22,CD25, CD30, CD31, CD33, CD34, CD40, CD44v6, CD45, CD51, CD52, CD54,CD56, CD64, CD70, CD74, CD79, CD105, CD117, CD123, CD133, CD138, CD144,CD146, CD152, CD174, CD205, CD227, CD326, CD340, Cripto, ED-B, GD2,TMEFF2, VEGFR1, VEGFR2, FGFR, PDGFR, ANGPT1, TIE1, TIE2, NRP1, TEK(CD202B), TGFβR, Death Receptor 5 (Trail-R2), DLL4, EPHA1, EPHA2, EPHA3,EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3,EPHB4, EPHB5, EPHB6, FAP, GPNMB, ICAMs, VCAMs, PSMA, HER-2/neu, IL-13Ralpha 2, MUC-1, MUC16, EGFR1 (HER-1), EGFR2 (HER-2/neu), EGFR3 (HER-3),IGF-1R, IGF-2R, c-Met (HGFR), Mesothelin, PDGFR, EDGR, TAG-72,transferrin receptor, EpCAM, CTLA-4, PSMA, tenascin C,alpha-fetoprotein, vimentin, C242 antigen, TRAIL-R1, TRAIL-R2, CA-125,GPNMB, CA-IX, GD3 ganglioside, RANKL, BAFF, IL-6R, TAG-72, HAMA, andCD166.

Examples of the molecular imaging agent include, but are not limited to,Gadolinium, ⁶⁴Cu diacetyl-bis(N⁴-methylthiosemicarbazone),¹⁸F-flourodeoxyglucose, ¹⁸F-flouride, 3′-deoxy-3′-[¹⁸F]fluorothymidine,¹⁸F-fluoromisonidazole, gallium, technetium-99, thallium, barium,gastrografin, iodine contrasting agents, iron oxide, green fluorescentprotein, luciferase, beta-galactosidase, and any combination of thepreceding.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted minicell vaccines against infectious disease agents in vivo,ex vivo, and/or in vitro and are used to prevent, inhibit, and/or slowthe progression of infectious disease agents in an animal by generatinga recipient animal host immune response that negatively impacts thedisease agent. In some embodiments, targeted minicells are (i) derivedfrom Fc-binding minicells wherein the Fc-binding minicells display theFc binding portion of Protein G on their surfaces, (ii) are loaded withone or more antigenic carbohydrates, protein antigens, and/or DNAvaccines any of which are derived from an infectious disease agent (iii)further comprise surface localized antibodies and/or Fc-containingfusion/conjugate molecules that are bound to the Fc binding portion ofthe surface displayed Fc binding region of Protein A wherein theantibodies and/or Fc-containing fusion/conjugate molecules recognize aeukaryotic antigen presenting cell-specific surface antigen and arecapable of stimulating receptor mediated endocytosis upon binding of thetargeted minicell to the eukaryotic antigen presenting cell-specificsurface antigen, (iv) further comprise an endosomal disrupting agent,including but not limited to LLO, (v) further comprise a general and/ortargeted molecular adjuvant, and (vi) further comprise apharmaceutically acceptable carrier for an in vivo route ofadministration including but not limited to intravenous, intramuscular,subcutaneous, intraperitoneal, oral, and/or nasal administration. Insome embodiments, targeted minicell vaccines elicit protective humoral(antibody-mediated) and/or cellular (cytotoxic T-cell mediated) immuneresponses in an animal. In one aspect, immune responses are protectiveagainst infectious disease agents including but not limited to agents ofbacterial, viral, and parasitic origin. In some embodiments, theantibody and/or Fc-containing fusion/conjugate molecule(s) on thesurface of the minicell vaccine recognizes but is not limited torecognizing one or more of CD11b, CD11c, DC-SIGN, CD8, DEC-205, CD105,Flt3, Flt3L, CD103, CD115, CD45, CX₃CR1, CCR7, SIRPa, CD205, DCIR2,CD40, M-CSFR, F4/80, CD123, and CD68. In some embodiments, the targetedmolecular adjuvant stimulates TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,TLR8, TLR9, TLR10, M-CSFR, and any combination of the preceding.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted minicell vaccines against infectious disease agents in vivo,ex vivo, and/or in vitro and are used to prevent, inhibit, and/or slowthe progression of infectious disease agents in an animal by generatinga recipient animal host immune response that negatively impacts thedisease agent. In this preferred embodiment targeted minicells are (i)derived from Fc-binding minicells wherein the Fc-binding minicellsdisplay the Fc binding portion of Protein A on their surfaces, (ii) areloaded with one or more antigenic carbohydrates, protein antigens,and/or DNA vaccines any of which are derived from an infectious diseaseagent (iii) further comprise surface localized antibodies and/orFc-containing fusion/conjugate molecules that are bound to the Fcbinding portion of the surface displayed Fc binding region of Protein Awherein the antibodies and/or Fc-containing fusion/conjugate moleculesrecognize a eukaryotic antigen presenting cell-specific surface antigenand are capable of stimulating receptor mediated endocytosis uponbinding of the targeted minicell to the eukaryotic antigen presentingcell-specific surface antigen, (iv) further comprise an endosomaldisrupting agent, including but not limited to LLO, (v) further comprisea general and/or targeted molecular adjuvant, and (vi) further comprisea pharmaceutically acceptable carrier for an in vivo route ofadministration including but not limited to intravenous, intramuscular,subcutaneous, intraperitoneal, oral, and/or nasal administration. Insome embodiments, targeted minicell vaccines elicit protective humoral(antibody-mediated) and/or cellular (cytotoxic T-cell mediated) immuneresponses in an animal. In some embodiments, immune responses areprotective against infectious disease agents including but not limitedto agents of bacterial, viral, and parasitic origin. In someembodiments, the antibody and/or Fc-containing fusion/conjugatemolecules on the surface of the minicell vaccine recognizes but is notlimited to recognizing one or more of CD11b, CD11c, DC-SIGN, CD8,DEC-205, CD105, Flt3, Flt3L, CD103, CD115, CD45, CX₃CR1, CCR7, SIRPa,CD205, DCIR2, CD40, M-CSFR, F4/80, CD123, and CD68. In some embodiments,the targeted molecular adjuvant stimulates TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, M-CSFR, and any combination of thepreceding.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted minicell vaccines against tumors in vivo, ex vivo, and/or invitro and are used to prevent, inhibit, and/or slow the progression oftumors in an animal by generating a recipient animal host immuneresponse that negatively impacts the tumor(s). In this preferredembodiment targeted minicells are (i) derived from Fc-binding minicellswherein the Fc-binding minicells display the Fc binding portion ofProtein G on their surfaces, (ii) are loaded with one or more antigeniccarbohydrates, protein antigens, and/or DNA vaccines any of which arederived from a tumor cell (iii) further comprise surface localizedantibodies and/or Fc-containing fusion/conjugate molecules that arebound to the Fc binding portion of the surface displayed Fc bindingregion of Protein A wherein the antibodies and/or Fc-containingfusion/conjugate molecules recognize a eukaryotic antigen presentingcell-specific surface antigen and are capable of stimulating receptormediated endocytosis upon binding of the targeted minicell to theeukaryotic antigen presenting cell-specific surface antigen, (iv)further comprise an endosomal disrupting agent, including but notlimited to cLLO, (v) further comprise a general and/or targetedmolecular adjuvant, and (vi) further comprise a pharmaceuticallyacceptable carrier for an in vivo route of administration including butnot limited to intravenous, intramuscular, subcutaneous,intraperitoneal, oral, and/or nasal administration. In some embodiments,targeted minicell vaccines elicit protective humoral (antibody-mediated)and/or cellular (cytotoxic T-cell mediated) immune responses in ananimal. In one aspect, immune responses are protective against malignantand/or benign tumors of autologous origin. In some embodiments, theantibody and/or Fc-containing fusion/conjugate molecule(s) on thesurface of the minicell vaccine recognizes but is not limited torecognizing one or more of CD11b, CD11c, DC-SIGN, CD8, DEC-205, CD105,Flt3, Flt3L, CD103, CD115, CD45, CX₃CR1, CCR7, SIRPa, CD205, DCIR2,CD40, M-CSFR, F4/80, CD123, and CD68. In some embodiments, the targetedmolecular adjuvant stimulates TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,TLR8, TLR9, TLR10, M-CSFR, and any combination of the preceding.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted minicell vaccines against tumors in vivo, ex vivo, and/or invitro and are used to prevent, inhibit, and/or slow the progression oftumors in an animal by generating a recipient animal host immuneresponse that negatively impacts the tumor(s). In this preferredembodiment targeted minicells are (i) derived from Fc-binding minicellswherein the Fc-binding minicells display the Fc binding portion ofProtein A on their surfaces, (ii) are loaded with one or more antigeniccarbohydrates, protein antigens, and/or DNA vaccines any of which arederived from a tumor cell (iii) further comprise surface localizedantibodies and/or Fc-containing fusion/conjugate molecules that arebound to the Fc binding portion of the surface displayed Fc bindingregion of Protein A, wherein the antibodies and/or Fc-containingfusion/conjugate molecules recognize a eukaryotic antigen presentingcell-specific surface antigen and are capable of stimulating receptormediated endocytosis upon binding of the targeted minicell to theeukaryotic antigen presenting cell-specific surface antigen, (iv)further comprise an endosomal disrupting agent, including but notlimited to LLO, (v) further comprise a general and/or targeted molecularadjuvant, and (vi) further comprise a pharmaceutically acceptablecarrier for an in vivo route of administration including but not limitedto intravenous, intramuscular, subcutaneous, intraperitoneal, oral,and/or nasal administration. In some embodiments, targeted minicellvaccines elicit protective humoral (antibody-mediated) and/or cellular(cytotoxic T-cell mediated) immune responses in an animal. In oneaspect, immune responses are protective against malignant and/or benigntumors. In some embodiments, the antibody and/or Fc-containingfusion/conjugate molecule(s) on the surface of the minicell vaccinerecognizes but is not limited to recognizing one or more of CD11b,CD11c, DC-SIGN, CD8, DEC-205, CD105, Flt3, Flt3L, CD103, CD115, CD45,CX₃CR1, CCR7, SIRPa, CD205, DCIR2, CD40, M-CSFR, F4/80, CD123, and CD68.In some embodiments, the targeted molecular adjuvant stimulates TLR1,TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, M-CSFR, and anycombination of the preceding.

In some preferred embodiments, targeted therapeutic minicells are usedas targeted minicell vaccines against tumors in vivo, ex vivo, and/or invitro and are used to prevent, inhibit, and/or slow the progression oftumors in an animal by generating a recipient animal host immuneresponse that negatively impacts the tumor(s). In some embodiments,targeted minicells are (i) derived from Fc-binding minicells wherein theFc-binding minicells display the Fc binding portion of Protein G ontheir surfaces, (ii) are loaded with one or more antigeniccarbohydrates, protein antigens, and/or DNA vaccines any of which arederived from a tumor cell (iii) further comprise surface localizedantibodies and/or Fc-containing fusion/conjugate molecules that arebound to the Fc binding portion of the surface displayed Fc bindingregion of Protein G, wherein the antibodies and/or Fc-containingfusion/conjugate molecules recognize a eukaryotic antigen presentingcell-specific surface antigen and are capable of stimulating receptormediated endocytosis upon binding of the targeted minicell to theeukaryotic antigen presenting cell-specific surface antigen, (iv)further comprise an endosomal disrupting agent, including but notlimited to LLO, (v) further comprise a general and/or targeted molecularadjuvant, and (vi) further comprise a pharmaceutically acceptablecarrier for an in vivo route of administration including but not limitedto intravenous, intramuscular, subcutaneous, intraperitoneal, oral,and/or nasal administration. In some embodiments, targeted minicellvaccines elicit protective humoral (antibody-mediated) and/or cellular(cytotoxic T-cell mediated) immune responses in an animal. In oneaspect, immune responses are protective against malignant and/or benigntumors. In some embodiments, the antibody and/or Fc-containingfusion/conjugate molecule(s) on the surface of the minicell vaccinerecognizes but is not limited to recognizing one or more of CD11b,CD11c, DC-SIGN, CD8, DEC-205, CD105, Flt3, Flt3L, CD103, CD115, CD45,CX₃CR1, CCR7, SIRPa, CD205, DCIR2, CD40, M-CSFR, F4/80, CD123, and CD68.In some embodiments, the targeted molecular adjuvant stimulates TLR1,TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, M-CSFR, and anycombination of the preceding.

In some preferred embodiments, the Fc-binding minicells described hereincan be used as analyte detection reagents for diagnostic assaysincluding but not limited to Lateral Flow Immunoassays (LFIAs). In someembodiments, the analyte-detecting Fc-binding minicells are comprised of(i) Fc-binding minicells that express and display the Fc-binding regionof Protein A, (ii) an analyte-specific antibody or otheranalyte-specific Fc-containing fusion/conjugate molecule bound to theFc-containing minicells, and (iii) a detection reagent including but notlimited to a small molecule flourophore, a fluorescent protein, anenzyme, a magnetic particle, and colloidal gold wherein the detectionreagent is encapsulated, displayed, or otherwise associated with theminicells. In a related permutation, Fc-binding minicells are used as anegative readout detection reagent for use in a competitive LFIA. Insome embodiments, negative readout Fc-binding minicells are comprised of(i) Fc-binding minicells, (ii) an Fc/analyte fusion/conjugate bound tothe Fc-containing minicells, and (iii) a detection reagent including butnot limited to a small molecule flourophore, a fluorescent protein, anenzyme, a magnetic particle, and colloidal gold wherein the detectionreagent is encapsulated, displayed, or otherwise associated with theminicells. Minicells are used as detection reagents in kits used toanalyze clinical, veterinary, environmental, solid and liquidfoodstuffs, pharmaceutical products, and drinking water for the presenceor absence of a given relevant analyte in solution. Lateral FlowImmunoassays are constructed whereby they contain (i) product backing,(ii) a sample pad, (iii) a particle conjugate pad, (iv) a porousmembrane (e.g. nitrocellulose), (v) a test line, (vi) a control line,and (vii) a wick material. LFIAs are used as rapid point-of-carediagnostics as well as for in-home use (e.g. pregnancy tests), andvarious field tests (e.g. determining toxin levels in drinking water orsoil). The product backing is selected from the group including but notlimited to polystyrene and/or another plastic polymer and may be coatedwith medium to high tack adhesive at the discretion of the artisan. Thesample pad is selected from material types including but not limited tocellulose, glass fiber, rayon, polypropylene, and/or other commonly usedfiltration media known in the art and at the discretion of the artisan.The particle conjugate pad is selected from material types including butnot limited to glass fiber, polyesters, polystyrene, polypropylene,rayons, and other filtration media known in the art at the discretion ofthe artisan. In addition, the conjugate pad component may be “blocked”by the addition by way of immersion into a solution containing proteins,polymers, surfactants, and any combination of the preceding. The porousanalytical test membrane is selected from material types includingnitrocellulose, nylon, polyvinylidene fluoride (PVDF), Fusion 5 matrix(Whatman), 4CastChip matrix (Amic, Uppsala, Sweden), and any combinationof the preceding. In addition, the porous analytical test membranecomponent may be “blocked” by the addition by way of immersion into asolution containing proteins, polymers, surfactants, and any combinationof the preceding. Blocking of the porous analytical membrane occursafter the test and control lines are incorporated into the porousanalytical membrane. The test and control lines are incorporated intothe porous analytical membrane by use of manufacturing equipmentincluding but not limited to non-contact pump-driven solenoiddispensers, contact tip dispensers, quantitative airbrush dispensers,and any combination of the preceding. Test strips are comprised of anantibody or other test analyte-specific binding partner that iscovalently or non-covalently linked to the porous analytical testmembrane. Control strips are comprised of an antibody or other detectionparticle-specific binding partner (e.g. an anti-minicell antibody) suchthat the detection particle (i.e. minicell) can be bound by the controlstrip independent of binding of the detection particle to the testanalyte. The wick component can be selected from material typesincluding but not limited to high-density cellulose. Many wickingcomponents are known to the artisan and can be utilized in the finalproduct at the discretion of the artisan. An illustrative embodiment ofa minicell-based Lateral Flow Immunoassay is depicted in FIG. 1. In someembodiments, test solutions are acquired in or are prepared in liquidsolution, applied to the sample pad, mix with the analyte detectionreagent (minicells), and then traverse the porous analytical membranetowards the wick. Analyte bound detection reagent accumulates at thepositive test line and may be detected using any number of methods knownin the art including photometric, charged coupled device camera,flourimetric analysis (e.g., LED excitation), radiometric analysis, andby Magnetic Assay Reader.

In some preferred embodiments, the Fc-binding minicells described hereinare used as analyte detection reagents for diagnostic assays includingbut not limited to Lateral Flow Immunoassays (LFIAs). In someembodiments, the analyte-detecting Fc-binding minicells are comprised of(i) Fc-binding minicells that express and display the Fc-binding regionof Protein G, (ii) an analyte-specific antibody or otheranalyte-specific Fc-containing fusion/conjugate molecule bound to theFc-containing minicells, and (iii) a detection reagent including but notlimited to a small molecule flourophore, a fluorescent protein, anenzyme, a magnetic particle, and colloidal gold wherein the detectionreagent is encapsulated, displayed, or otherwise associated with theminicells. In a related permutation, Fc-binding minicells are used as anegative readout detection reagent for use in a competitive LFIA. Insome embodiments, negative readout Fc-binding minicells are comprised of(i) Fc-binding minicells, (ii) an Fc/analyte fusion/conjugate bound tothe Fc-containing minicells, and (iii) a detection reagent including butnot limited to a small molecule flourophore, a fluorescent protein, anenzyme, a magnetic particle, and colloidal gold wherein the detectionreagent is encapsulated, displayed, or otherwise associated with theminicells. Minicells are used as detection reagents in kits used toanalyze clinical, veterinary, environmental, solid and liquidfoodstuffs, pharmaceutical products, and drinking water for the presenceor absence of a given relevant analyte in solution. Lateral FlowImmunoassays are constructed whereby they contain (i) product backing,(ii) a sample pad, (iii) a particle conjugate pad, (iv) a porousmembrane (e.g. nitrocellulose), (v) a test line, (vi) a control line,and (vii) a wick material. LFIAs are used as rapid point-of-carediagnostics as well as for in-home use (e.g. pregnancy tests), andvarious field tests (e.g. determining toxin levels in drinking water orsoil). The product backing is selected from the group including but notlimited to polystyrene and/or another plastic polymer and may be coatedwith medium to high tack adhesive at the discretion of the artisan. Thesample pad is selected from material types including but not limited tocellulose, glass fiber, rayon, polypropylene, and/or other commonly usedfiltration media known in the art and at the discretion of the artisan.The particle conjugate pad is selected from material types including butnot limited to glass fiber, polyesters, polystyrene, polypropylene,rayons, and other filtration media known in the art at the discretion ofthe artisan. In addition, the conjugate pad component may be “blocked”by the addition by way of immersion into a solution containing proteins,polymers, surfactants, and any combination of the preceding. The porousanalytical test membrane is selected from material types includingnitrocellulose, nylon, polyvinylidene fluoride (PVDF), Fusion 5 matrix(Whatman), 4CastChip matrix (Amic, Uppsala, Sweden), and any combinationof the preceding. In addition, the porous analytical test membranecomponent may be “blocked” by the addition by way of immersion into asolution containing proteins, polymers, surfactants, and any combinationof the preceding. Blocking of the porous analytical membrane occursafter the test and control lines are incorporated into the porousanalytical membrane. The test and control lines are incorporated intothe porous analytical membrane by use of manufacturing equipmentincluding but not limited to non-contact pump-driven solenoiddispensers, contact tip dispensers, quantitative airbrush dispensers,and any combination of the preceding. Test strips are comprised of anantibody or other test analyte-specific binding partner that iscovalently or non-covalently linked to the porous analytical testmembrane. Control strips are comprised of an antibody or other detectionparticle-specific binding partner (e.g. an anti-minicell antibody) suchthat the detection particle (i.e. minicell) can be bound by the controlstrip independent of binding of the detection particle to the testanalyte. The wick component may be selected from material typesincluding but not limited to high-density cellulose. Many wickingcomponents are known to the artisan and can be utilized in the finalproduct at the discretion of the artisan. A complete diagram of aminicell-based Lateral Flow Immunoassay is depicted in FIG. 1. In someembodiments, test solutions are acquired in or are prepared in liquidsolution, applied to the sample pad, mix with the analyte detectionreagent (minicells), and then traverse the porous analytical membranetowards the wick. Analyte bound detection reagent accumulates at thepositive test line and may be detected using any number of methods knownin the art including photometric, charged coupled device camera,flourimetric analysis (e.g. LED excitation), radiometric analysis, andby Magnetic Assay Reader.

Some embodiments provide an Fc-binding minicell-producing bacteriumcomprising: (i) an expressible gene encoding a minicell-producing geneproduct that modulates one or more of septum formation, binary fission,and chromosome segregation; (ii) an expressible “genetic suicide” geneencoding a heterologous endonuclease, where the chromosome of theminicell-producing bacteria comprises one or more recognition sites ofthe endonuclease; (iii) a defined auxotrophy; (iv) a deletion ormutation in the lpxM/msbB gene (or other functional equivalent); and (v)a recombinant expression cassette capable of the functional expressionand surface display of the Fc binding region of Protein G. In someembodiments, the minicell-producing gene is a cell division gene. Thecell division gene includes, but is not limited to ftsZ, sulA, ccdB, andsfiC. In some embodiments, the mincell-producing gene is expressed underthe control of an inducible promoter. In some embodiments, theendonuclease suicide gene is located on the chromosome of theminicell-producing bacteria. In some embodiments, the endonuclease is ahoming endonuclease. The homing endonuclease includes, but is notlimited to, I-CeuI, PI-SceI, I-ChuI, I-CpaI, I-SceIII, I-CreI, I-MsoI,I-SceII, I-SceIV, I-CsmI, I-DmoI, I-PorI, PI-TliI, PI-TliII, andPI-ScpI. In some embodiments, the endonuclease is expressed under thecontrol of an inducible promoter. In some embodiments, the auxotrophy isdue to a deletion or inactivating mutation in an essential metabolicgene. In some embodiments the deletion or inactivating mutation is inthe dapA gene or its functional homolog. In some embodiments, theminicell-producing bacteria further comprises a deletion or aninactivating mutation in a gene encoding a gene product that is involvedin lipopolysaccharide synthesis, wherein the gene is geneticallymodified compared to a corresponding wild-type gene. In someembodiments, the inactivated gene is lpxM/msbB which encodes a geneproduct that causes the bacteria to produce an altered lipid A moleculecompared to lipid A molecules in a corresponding wild-type bacterium. Insome embodiments, the altered lipid A molecule is deficient with respectto the addition of myristolic acid to the lipid A portion of thelipopolysaccharide molecule compared to lipid A molecules in acorresponding wild-type bacterium. In some embodiments, theminicell-producing bacteria further comprise a deletion or inactivatingmutation in a gene that is involved in homologous recombination, wherethe gene is genetically modified compared to a corresponding wild-typegene, where the minicell-producing bacteria are deficient in DNA damagerepair. In some embodiments, the minicell-producing bacteria furthercomprise a mutation in or lack the gene coding for ribonuclease III(e.g., E. coli's rnc gene; degrades double-stranded RNAs in E. coli)such that the resulting minicells are deficient in this ribonucleasethereby increasing the half-life of double-stranded RNA molecules,including siRNA and shRNA in minicells. In some embodiments theminicell-producing bacterial strain further comprises a recombinant cLLOprotein such that resulting minicells further comprise the cLLO protein.In some embodiments the Fc-binding minicell-producing bacterium is aGram-negative bacterium including but not limited to Campylobacterjejuni, Haemophilus influenzae, Bordetella pertussis, Brucella spp.,Franciscella tularemia, Legionella pneumophilia, Neisseria meningitidis,Kliebsella, Yersinia spp., Helicobacter pylori, Neisseria gonorrhoeae,Legionella pneumophila, Salmonella spp., Shigella spp., Pseudomonasaeruginosa, and Escherichia coli. In some embodiments the Fc-bindingminicell-producing bacterium is a Gram-positive bacterium including butnot limited to Staphylococcus spp., Lactobacillus spp., Streptococcusspp., Bacillus subtilis, Clostridium difficile, and Bacillus cereus.

Some embodiments provide an Fc-binding minicell-producing bacteriumcomprising: (i) an expressible gene encoding a minicell-producing geneproduct that modulates one or more of septum formation, binary fission,and chromosome segregation; (ii) an expressible “genetic suicide” geneencoding a heterologous endonuclease, where the chromosome of theminicell-producing bacteria comprises one or more recognition sites ofthe endonuclease; (iii) a defined auxotrophy; (iv) a deletion ormutation in the lpxM/msbB gene (or other functional equivalent); and (v)a recombinant expression cassette capable of the functional expressionand surface display of the Fc binding region of Protein A. In someembodiments, the minicell-producing gene is a cell division gene. Thecell division gene includes, but is not limited to ftsZ, sulA, ccdB, andsfiC. In some embodiments, the mincell-producing gene is expressed underthe control of an inducible promoter. In some embodiments, theendonuclease suicide gene is located on the chromosome of theminicell-producing bacteria. In some embodiments, the endonuclease is ahoming endonuclease. The homing endonuclease includes, but is notlimited to, I-CeuI, PI-SceI, I-ChuI, I-CpaI, I-SceIII, I-CreI, I-MsoI,I-SceII, I-SceIV, I-CsmI, I-DmoI, I-PorI, PI-TliI, PI-TliII, andPI-ScpI. In some embodiments, the endonuclease is expressed under thecontrol of an inducible promoter. In some embodiments, the auxotrophy isdue to a deletion or inactivating mutation in an essential metabolicgene. In some embodiments the deletion or inactivating mutation is inthe dapA gene or its functional homolog. In some embodiments, theminicell-producing bacteria further comprises a deletion or aninactivating mutation in a gene encoding a gene product that is involvedin lipopolysaccharide synthesis, wherein the gene is geneticallymodified compared to a corresponding wild-type gene. In someembodiments, the inactivated gene is lpxM/msbB which encodes a geneproduct that causes the bacteria to produce an altered lipid A moleculecompared to lipid A molecules in a corresponding wild-type bacterium. Insome embodiments, the altered lipid A molecule is deficient with respectto the addition of myristolic acid to the lipid A portion of thelipopolysaccharide molecule compared to lipid A molecules in acorresponding wild-type bacterium. In some embodiments, theminicell-producing bacteria further comprise a deletion or inactivatingmutation in a gene that is involved in homologous recombination, wherethe gene is genetically modified compared to a corresponding wild-typegene, where the minicell-producing bacteria are deficient in DNA damagerepair. In some embodiments, the minicell-producing bacteria furthercomprise a mutation in or lack the gene coding for ribonuclease III(e.g., E. coli's mc gene; degrades double-stranded RNAs in E. coli) suchthat the resulting minicells are deficient in this ribonuclease therebyincreasing the half-life of double-stranded RNA molecules, includingsiRNA and shRNA in minicells. In some embodiments the minicell-producingbacterial strain further comprises a recombinant cLLO protein such thatresulting minicells further comprise the cLLO protein. In someembodiments the Fc-binding minicell-producing bacterium is aGram-negative bacterium including but not limited to Campylobacterjejuni, Haemophilus influenzae, Bordetella pertussis, Brucella spp.,Franciscella tularemia, Legionella pneumophilia, Neisseria meningitidis,Kliebsella, Yersinia spp., Helicobacter pylori, Neisseria gonorrhoeae,Legionella pneumophila, Salmonella spp., Shigella spp., Pseudomonasaeruginosa, and Escherichia coli. In some embodiments the Fc-bindingminicell-producing bacterium is a Gram-positive bacterium including butnot limited to Staphylococcus spp., Lactobacillus spp., Streptococcusspp., Bacillus subtilis, Clostridium difficile, and Bacillus cereus.

Some embodiments provide a method of making Fc-binding minicells,comprising culturing the Fc-binding minicell-producing bacteriadisclosed herein and substantially separating minicells from theminicell-producing parent cells, thereby generating a compositioncomprising Fc-binding minicells. In some embodiments, the method furthercomprises inducing minicell formation from the minicell-producing parentcell. In some embodiments, the method further comprises inducingexpression of the gene encoding the genetic suicide endonuclease. Insome embodiments, minicell formation is induced by the presence of oneor more chemical compounds selected from isopropylβ-D-1-thiogalactopyranoside (IPTG), rhamnose, arabinose, xylose,fructose, melibiose, and tetracycline. In some embodiments, theexpression of the gene encoding the genetic suicide endonuclease isinduced by a change in temperature. In some embodiments, the methodfurther comprises purifying the Fc-binding minicells from thecomposition. In some embodiments, the minicells are substantiallyseparated from the parent cells by a process selected from the groupincluding but not limited to centrifugation, filtration,ultrafiltration, ultracentrifugation, density gradation, immunoaffinity,immunoprecipitation, and any combination of the preceding purificationmethods.

Some embodiments provide a eubacterial minicell comprising an outermembrane, where the lipopolysaccharide constituents of the outermembrane comprises Lipid A molecules having no myristolic acid moiety(“detoxified lipopolysaccharide” or “detoxified LPS”). Detoxified LPSresults in the reduction of pro-inflammatory immune responses in amammalian host compared to the inflammatory response induced by theouter membrane of eubacterial minicells that are derived from acorresponding wild-type bacterium.

In some embodiments, the targeted therapeutic minicell further comprisesone or more biologically active compounds. In some embodiments, at leastone of the biologically active compounds is selected from the groupconsisting of a radioisotope, a polypeptide, a nucleic acid, and a smallmolecule drug. The biologically active compound(s) are selected from thegroup including but not limited to therapeutic nucleic acid(s), smallmolecule drug(s), pro-drug(s), therapeutic polypeptide(s), smallmolecule imaging agent(s), protein-based imaging agent(s), a eukaryoticexpression plasmid encoding for protein-based imaging agent(s), pro-drugconverting enzyme(s) and any combination of the preceding. Thebiologically active compound can also be a combination of a nucleic acidand a small molecule; a combination of a small molecule imaging agentand a small molecule drug; a combination of a small molecule drug, asmall molecule imaging agent, and a nucleic acid; or a combination of anucleic acid and a polypeptide.

The present application describes a composition comprising Fc-bindingeubacterial minicells capable of binding and displaying antibodiesand/or Fc-containing fusion/conjugate molecules to facilitate theminicell-based targeted delivery of several classes of bioactive payloadin concert or singular wherein the final preparation of targetedminicells is sufficiently devoid of remaining viable contaminatingparent cells. The minicells may or may not further comprise a detoxifiedform of lipopolysaccharide at the option and discretion of the artisan.

1. Minicell Production

Minicells are achromosomal, membrane-encapsulated biologicalnano-particles (approximately 250-500 nm in diameter depending on thestrain type and growth conditions used) that are formed by bacteriafollowing a disruption in the normal cell division apparatus. Inessence, minicells are small, metabolically active replicas of normalbacterial cells with the exception that they contain no chromosomal DNAand as such, are non-dividing and non-viable. Although minicells do notcontain chromosomal DNA, plasmid DNA, RNA, native and/or recombinantlyexpressed proteins, and other metabolites have all been shown tosegregate into minicells.

Disruptions in the coordination between chromosome replication and celldivision lead to minicell formation from the polar region of mostrod-shaped prokaryotes. Disruption of the coordination betweenchromosome replication and cell division can be facilitated through theover-expression of some of the genes involved in septum formation andbinary fission. Alternatively, minicells can be produced in strains thatharbor mutations in genes involved in septum formation and binaryfission. Impaired chromosome segregation mechanisms can also lead tominicell formation as has been shown in many different prokaryotes.

Similarly, minicell production can be achieved by the over-expression ormutation of genes involved in the segregation of nascent chromosomesinto daughter cells. For example, mutations in the parC or mukB loci ofE. coli have been demonstrated to produce minicells. Both affectseparate requisite steps in the chromosome segregation process inEnterobacteriacea. It can be assumed that like the cell division genesdescribed above, manipulation of wild type levels of any given geneinvolved in the chromosome segregation process that result in minicellproduction will have similar effects in other family members.

Because the cell division and chromosome replication processes are socritical to survival, there exists a high level of genetic andfunctional conservation amongst prokaryotic family members with respectto genes responsible for these processes. As a result, theover-expression or mutation of a cell division gene capable of drivingminicell production in one family member, can be used to produceminicells in another. For example, it has been shown that theover-expression of the E. coli ftsZ gene in other Enterobacteriaceafamily members such as Salmonella spp. and Shigella spp as well as otherclass members such as Pseudomonas spp. will result in similar levels ofminicell production.

The same can be demonstrated in the mutation-based minicell producingstrains of the family Enterobacteriacea. For example, deletion of themin locus in any of Enterobacteriacea family members results in minicellproduction. Cell division genes from the Enterobacteriacea in whichmutation can lead to minicell formation include but are not limited tothe min genes (MinCDE). While minicell production from the min mutantstrains is possible, these strains have limited commercial value interms of being production strains. The reason for this is that strainswith deletions or mutations within the min genes make minicells atconstitutively low levels. This presents two problems in terms ofcommercialization and economies of scale. The first is that minicellyields from these strains are low, which increases production cost. Thesecond is that minicell yields are highly variable with the mutantstrains and lot-to-lot variability has an enormous impact on productioncost, manufacturing quality control and regulatory compliance. Usingcell division mutant strains to produce minicells that encapsulatebiologically active molecules such as proteins, RNA, DNA, and othercatabolites for diagnostic or therapeutic delivery is problematic. Theonset of minicell production in the mutant strains cannot be controlledand occurs at a low level so that the end result is that some minicellswill contain no biologically active molecules while others will containwidely variable amounts of biologically active molecules. Theseshortcomings when taken together or separately greatly restrict theutility of these mutant strains for commercial purposes.

Minicell-producing strains that overexpress cell division genes(“overexpressers”) are preferred over mutation-based strains because theminicell-production phenotype is controllable as long as the celldivision genes to be overexpressed are placed under the control of aninducible or other conditionally active eubacterial promoter system.Minicell production from strains overexpres sing the cell division geneftsZ were discovered by researchers who were identifying essential celldivision genes in E. coli using plasmid-based complementation studies.In these studies, the ftsZ gene was present in over 10 copies per cell.The presence of multiple gene copies of ftsZ was demonstrated to produceminicells and extremely long filamented cells. Ultimately, thistransition into the irreversible filamentous phenotype negativelyimpacts minicell yields from strains overexpressing ftsZ from multi-copyplasmids, although the number of minicells produced is still higher thanthat of any mutant strain. It has since been demonstrated that byreducing the number of ftsZ gene copies to a single, chromosomalduplication, the number of minicells produced increases over thosestrains where ftsZ is located on multi-copy plasmids and that thefilamentous phenotype is less profound. Thus, the preferredcomposition(s) are minicell-producing strains that inducibly overexpressthe ftsZ gene from a duplicate, chromosomally integrated copy of ftsZ.The duplicate ftsZ gene used can be derived directly from the species ofbacteria in which the minicell-production phenotype is being engineeredand can also be derived from the ftsZ gene sequence from other speciesof bacteria. By way of non-limiting example, overexpression of the ftsZgene of Escherichia coli can be used to generate minicells fromEscherichia coli and Salmonella typhimurium. Resulting strains arecomprised of the wild type ftsZ gene and a separate, duplicative, andinducible copy of the ftsZ gene on the chromosome and the induciblegenetic suicide mechanism(s) described in U.S. patent publication No.2010/0112670, which is incorporated herein by its entirety. By way ofnon-limiting example, division genes that can be over-expressed toproduce minicells in the family Enterobacteriaceae include but are notlimited to ftsZ, minE, sulA, ccdB, and sfiC. The preferred compositionis to have a duplicate copy(s) of a cell division gene(s) under thecontrol of an inducible promoter that is stably integrated into thechromosome of a given eubacterial strain. It is easily recognized by oneskilled in the art that this same strategy could be imparted if theinducible cell division gene cassette were present on a plasmid, cosmid,bacterial artificial chromosome (BAC), recombinant bacteriophage orother episomal DNA molecule present in the cell.

This inducible phenotype approach to minicell production has severaldistinct advantages over the mutant systems. The first is that becausethere are no constitutive genetic mutations in these strains, thereexists no selective pressure during normal growth and the cells of theculture maintain a very stable and normal physiology until the minicellphenotype is induced. The end result is that inducible minicellproducing strains are healthier and more stable, which ultimatelyresults in higher yields of minicells. Another distinct advantage ofusing the inducible phenotype approach to minicell production is incases where minicells are to be used to deliver biologically activemolecules such as proteins, therapeutic RNAs, plasmid DNAs, and otherbioactive catabolites that can be made by the minicell-producing parentcells such that the minicells that are produced encapsulate thosebiologically active molecules. In these cases, the preferred method isto induce the formation of the biologically active molecule(s) withinthe parental cells prior to inducing the minicell phenotype, so that allof the minicells produced will contain the desired amount of thebiologically active molecule(s). Alternatively, the minicells themselvesare capable of producing the bioactive molecule after being separatedfrom the parental cells. This includes but is not limited to forming thebioactive molecule from an episomal nucleic acid or RNA encoding for thebioactive molecule located within the minicell or by preexisting proteinconstituents of minicells after being separated from the parental cells.Any of these expression strategies can be employed to express anddisplay binding moieties on the surfaces of minicells. These advantages,when used in combination, result in a higher quality and quantity ofminicells. In addition, these minicells can further comprise smallmolecule drugs that can be loaded into minicells as described in moredetail below.

2. Minicell Purification

Because minicells are derived from some bacteria that are pathogenic oropportunistically pathogenic, it is of the utmost importance that anycontaminating parental cells be functionally eliminated from a givenpopulation before administration. Conventionally, live parental cellshave been eliminated through either physical means or biological meansor both.

Physical means include the use of centrifugation-based separationprocedures, filtration methodologies, chromatography methodologies, orany combination thereof.

Biological elimination is achieved by but not limited to thepreferential lysis of parental cells, the use of auxotrophic parentalstrains, treatment with antibiotics, treatment with UV radiation,diaminopimelic acid (DAP) deprivation, selective adsorption of parentalcells, treatment with other DNA damaging agents, and induction of asuicide gene.

Preferential lysis of parental cells is typically mediated by inducingthe lytic cycle of a lysogenic prophage. In the case of minicellproducing strains, it is most useful to use a prophage that is lysiscompetent but defective at re-infection, such that minicells are notsubsequently infected and lysed during activation of the lyticphenotype. Alternatively and by way of non-limiting example, individualgenes such as those classified as members of the holin gene family, canbe expressed to achieve similar levels of lysis without the concernsover re-infection inherent to the use of lysogenic prophages. Bothapproaches are limited by the fact that the lysis event, regardless ofthe method used to achieve it, expels unacceptable amounts of freeendotoxin into the media. Removal of such large amounts of freeendotoxin is time consuming, suffers from lot to lot variability, and isultimately cost prohibitive.

The use of auxotrophic strains raises concerns over reversion and assuch can only be used in cases where minicells are to be produced fromcommensal or non-pathogenic strains of bacteria. Thus, their applicationis limited with respect to being used as a method for elimination oflive non-pathogenic parental cells used in minicell production.

The use of antibiotics can be of benefit in the production of minicellswhen used on samples that have been enriched for minicells (bydifferential centrifugation or preliminary filtration for example). Withmany fewer parental cells present, the potential for the development ofantibiotic resistance is reduced to nearly zero. The use of antibioticson primary minicell production cultures that still contain high numbersof viable parental cells is undesirable as the chances for thedevelopment of antibiotic resistance increases proportionally to thenumber of viable parental cells.

Treatment with UV irradiation can be useful in the elimination of liveparental cells on a minicell production run with the exception of thefact that UV irradiation is random with respect to its effects onnucleic acids and results are highly variable from lot to lot. Inaddition, this method is not preferred when using minicells to delivertherapeutic or prophylactic nucleic acids as UV irradiation randomlydamages all nucleic acids. For instance, plasmid DNA would also behighly susceptible to DNA damage by UV irradiation and may be renderedineffective although still effectively delivered by minicells.

Diaminopimelic acid (DAP) deprivation can be useful in the eliminationof live parental cells with the exception that this approach is limitedby the number of species it can be used for. In other words, not allparent cell species capable of producing minicells require DAP forsurvival. DAP mutants in E. coli minicell-producing strains are of greatadvantage and in some cases preferred over the wild type. The advantageof using DAP is that this compound (diaminopimelic acid, an E. coli cellwall constituent) is critical for the growth of E. coli and is notpresent in or produced by animals. Thus, should a “viable” E. coliminicell-producing parental cell be administered along with targetedminicells, the parental cell will be unable to grow and will thereby beinert to the animal and with respect to minicell activity. A similarapproach can be used with Salmonella spp. based minicell-producingparental strains except in that case the aro genes, preferably aroB areremoved.

Selective adsorption methodologies have yet to be explored with respectto purifying minicells from viable parental cells. Selective adsorptionis defined as any process by which parental cells or minicells arepreferentially adsorbed to a substrate by virtue of their affinity forthe substrate. By way of non-limiting example, high affinityprotein-protein interactions could be exploited for this use. By way ofnon-limiting example, the outer membrane protein Invasin from thegram-negative species Yersinia pseudotuberculosis has a high affinityfor mammalian integrins. The gene encoding for invasin under the controlan inducible promoter could easily be introduced on to the chromosome ofa minicell producing strain. Minicells could be produced from thisstrain prior to the activation of expression of the invasin gene suchthat the minicells produced do not express or display invasin on theircell surface. Once the desired quantity of minicells is produced fromthe strain, the viable cells within the culture could be given thesignal to produce the invasin protein such that invasin is onlyexpressed and displayed upon viable cells. Once invasin ispreferentially expressed on the surface of viable parental cells, theycan be easily adsorbed to a substrate coated with integrins or otherinvasin-specific protein binding motifs embedded into a syntheticpolypeptide or other recombinant protein. Once absorbed, minicells canbe selectively purified away from viable parental cells by a number ofdifferent means dependent upon the substrate type used. Substratesinclude but are not limited to solid-phase chromatographic columns usedin gravity filtration applications, magnetic beads, ion exchangecolumns, or HPLC columns. This approach is limited by the disadvantagethat no single protein-protein interaction will work for all species ofminicell producing parent cells. For instance, the invasin-integrinapproach described above would be useful for most Gram-negativeEnterobacteriacea family members but not for use with minicell producingGram-positive Bacillaceae family members.

In some embodiments, minicells are substantially separated from theminicell-producing parent cells in a composition comprising minicells.For example, after separation, the composition comprising the minicellsis less than about 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%,77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35% or 30% free of minicell-producing parent cells. In some embodiments,the composition contains less than about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% minicell-producingparent cells.

Preferably, the final composition contains few enough contaminatingparental cells, viable or otherwise, so as not to be too toxic orinterfere with the activity of targeted minicells when administered invivo for therapeutic purposes.

Some preferred method of sufficiently eliminating contaminating viableparental bacterial cells is through the incorporation and activation ofan inducible genetic suicide mechanism, including but not limited to theactivation and expression of a homing endonuclease or functionalequivalent thereof as described in U.S. Patent Publication No.20100112670 prior to further physical separation methodologies such asstandard filtration techniques known in the art.

3. Targeting Minicells to Specific Cells, Tissues, and Organs

Following production, activation of the genetic suicide mechanism, andsubsequent purification, minicells are used as targeted deliveryvehicles. Minicells expressing the Fc binding region of Protein G orProtein A and further displaying antibodies, Fc-containing antibodyderivatives, and/or Fc-containing fusion/conjugate targeting moleculeson their surfaces are used to target specific cell types involved indisease in vivo to preferentially deliver their bioactive payloads tothe targeted tissue, organ, and cell type.

Antibodies, or any portion thereof, intended to aid in the targeting ofminicells to a specific tissue, organ, and cell type involved in diseasecan be derived from or be part of any immunoglobulin subclass, includingbut not limited to IgA, IgM, IgD, IgG, or IgE. Antibodies of anysubclass intended for facilitating the targeting function of minicellscan be “humanized”, although any antibody of any subclass against a cellspecific antigen can be raised in any animal known to generate antibodyresponses through adaptive immunity to achieve the same goal. In nature,antibodies are generated such that they contain two separate arms (Fab's), each of which recognizes the same epitope of a particular antigen.

In the laboratory, antibodies can be engineered to be independentlyspecific for different antigens, such that a single antibody targets twoseparate antigens simultaneously. By way of non-limiting example,antibodies can be engineered to recognize putative surface components ofa given eubacterial minicell (e.g., LPS O-antigens) on one arm and theother arm be engineered to recognize a eukaryotic cell-specific surfaceantigen such as those listed above. Additionally, those skilled in theart readily recognize that other bi-specific antibody approaches may beimplemented to achieve the same effect. By way of non-limiting example,one skilled in the art would readily recognize that two separateantibodies, with separate specificities, can be non-covalently attachedby coupling them to Protein A/G to form a crude “bi-specific” antibodyderivative capable of adhering to the surface of minicells wherein oneantibody within the complex specifically adheres to the surface of theminicell and the other antibody is displayed to specifically recognizeand thereby “target” a specific cell, tissue, or organ type involved indisease in vivo. Similarly, one skilled in the art will recognize thattwo separate antibodies, with separate specificities, could becovalently linked using various cross-linking techniques to achieve thesame effect. All of these potential approaches to targeting are readilyrecognized by those skilled in the art.

In some embodiments, other non-antibody based targeting approaches thatare collectively based on Fc-containing fusions/conjugates can be used.Examples of molecular targeting moiety that can be used, including butnot limited to receptor ligands, polypeptides, hormones, carbohydrates,aptamers, antibody-like molecules, and DARPins. Fc-conjugation may beachieved using a variety of approaches known in the art. In the case ofFc-containing polypeptide fusions, including but not limited to receptorligand/Fc fusions, Fc-containing peptide fusions, and Fc-containingDARPins, recombinant expression of the fusion is the preferred method ofconstruction. In the recombinant expression context, Fc regions may befused to either the amino or carboxy terminus of a given recombinantfusion at the discretion of the artisan such that fusion to the Fcregion does not affect ligand activity with respect to receptor bindingand stimulation of receptor-mediated endocytosis. Another approach tomaking Fc-containing polypeptides, peptides, and DARPins is by chemicalconjugation (a.k.a. cross-linking) of purified recombinant Fc regionmolecules to recombinant polypeptide, peptide, and/or DARPin moleculesusing any of the well known cross-linking techniques known in the art.In the context of chemical cross-linking, it is advantageous to include“reactive” amino acid groups on either or both of the purifiedrecombinant Fc-region or the polypeptide, peptide, and/or DARPinmolecule to be conjugated. Reactive amino acids typically include butare not limited to those that contain sulfhydryl groups, preferably acysteine residue. For use with popular heterobifunctional cross-linkingreagents, it is preferable to include a lysine residue at the linkagesite of the opposing conjugate (e.g. Fc-region contains a cysteineresidue while polypeptide contains a lysine or vice versa). In instanceswhere purified recombinant Fc regions are cross-linked to hormones,carbohydrates, aptamers, and other non amino acid and/or peptide basedmolecules, the skilled artisan will recognize that many othercross-linking reagents can be employed to achieve the same.Cross-linking reagents can be “homobifunctional” or “heterobifunctional”(having the same or different reactive groups, respectively) Examples ofcross-linking reagents include, but are not limited to, those listed inTable 1. Table 1 also illustrates which cross-linking reagents areappropriate and preferable for each conjugate molecule type/approach. Inutilizing this approach, construction and administration of the targetedtherapeutic minicells can be achieved by (i) producing or purchasingrecombinant Fc region, (ii) producing or purchasing the targetingmolecule to be conjugated to the Fc region, (iii) mixing the recombinantFc region with the targeting molecule in the presence of the appropriatecross-linking reagent (see Table 1) and incubating the mixture underconditions that will allow cross-linking to occur, (iv) purifyingresulting Fc-containing conjugates away from the reaction mixturefollowed by quantification of the Fc-containing conjugates, (v)incubating payload-containing minicells with an amount of Fc-containingconjugate sufficient enough to occupy all Fc-binding sites on thesurface of the minicells in the appropriate binding buffer, (vi)removing any unbound conjugates by any one or more conventional means(e.g., tangential flow filtration), (vii) concentrating and/orlyophilizing targeted therapeutic minicells, (v) formulating for productadministration the targeted therapeutic minicells by reconstituting inan appropriate volume of a pharmaceutically acceptable carrier.

The minicells described herein are genetically engineered to express anddisplay the Fc binding region of Protein G or Protein A on theirsurfaces. A preferred method to achieve expression and surface displayof the Fc region of Protein G or Protein A is by fusion of the Fcbinding region with an outer membrane of the “autotransporter” family.The monomeric autotransporters belonging to the sub-class type 5secretion system of autotransporters (commonly classified as type 5a)are most preferred. Included in that family of autotransportersclassified as type 5a, is the IgA protease (IgAP) of Neisseriagonorrhoeae. The IgAP autotransporter passenger domain is easilyreplaced by the Fc binding region of Protein G or Protein A. Severaldifferent antibody fragments and antibody fragment types have beendisplayed and characterized using the IgAP system in E. coli althoughthis approach is entirely novel with respect to its use to express anddisplay the Fc binding region of Protein G or Protein A to produceFc-binding minicells. The adhesin-involved-in-diffuse-adherance (AIDA-I)autotransporter from E. coli can also be used for display of Fc bindingregions of Protein G or Protein A. Once the Fc-binding minicells arebound to the antibody and/or Fc containing fusion/conjugate targetingmolecule, they become targeting-competent and are capable ofpreferentially localizing and accumulating in target tissues, organs, orcell types.

Some preferred embodiments for displaying the Fc region of Protein G orProtein A on the surface of minicells include the use of fusion withLpp-OmpA (SEQ ID NO 22 and SEQ ID NO:23, respectively). OmpA is an outermembrane protein of Escherichia coli that when fused to a lipoproteinleader sequence and a display protein of interest, can be exported tothe surface of E. coli. Because E. coli minicells are derived fromparental E. coli, Lpp-OmpA fusion proteins will be localized on thesurface of minicells as well. Several different antibody fragments andantibody fragment types have been displayed and characterized using theLpp-OmpA system in E. coli although this approach is entirely novel withrespect to its use to express and display the Fc binding region ofProtein G or Protein A to produce Fc-binding minicells. Once theFc-binding minicells are bound to the antibody and/or Fc-containingfusion/conjugate targeting molecule, they become targeting-competent andare capable of preferentially localizing and accumulating in targettissues, organs, or cell types.

Other native outer membrane proteins that can be used as fusion partnersto express and display one or more of the Fc binding regions of ProteinG or Protein A on the surface of minicells include but are not limitedto LamB, OmpF, OmpC, OmpD, PhoE, PAL, Type III secretion systems, pilusproteins, bacterial autotransporter protein family members, and variousflagellin proteins. Generally, the same approach could be used toexpress and display one or more of the Fc binding regions of Protein Gor Protein A on the surface of minicells derived from anyEnterobacteriacea or Bacillaceae family member such that the minicellsbecome Fc-binding minicells capable of further binding antibodies and/orFc-containing fusion/conjugate targeting molecules specific foreukaryotic cell-specific surface antigens, thereby becomingtargeting-competent minicells capable of preferentially localizing andaccumulating in target tissues, organs, or cell types involved indisease. One skilled in the art will recognize that achieving this goalis a matter of creating a nucleic acid sequence encoding a fusionprotein between a putative or predicted outer membrane protein or outermembrane localization sequence and one or more of the Fc binding regionsof Protein G or Protein A.

Fc-binding minicells bind antibodies and/or Fc-containingfusion/conjugate targeting molecules that are specific for cell-specificsurface antigens to become targeting-competent minicells.Targeting-competent minicells are further loaded with and/orrecombinantly express and encapsulate a bioactive payload including butnot limited to small molecule drugs, bioactive nucleic acids, bioactiveproteins, bioactive radionuclides, imaging agents, and bioactivelipopolysaccharides, and any combination of the proceeding to produce a“biological effect” (synonymous with biological response) thatnegatively impacts diseased cells, tissues, or organs or positivelyeffects the production of signals that indirectly mitigate diseased,cells, tissues, or organs in an animal. Targeting-competent minicellsare made to target eukaryotic cell-specific surface antigens of choiceincluding, but not limited to including α3β1 integrin, α4β1 integrin,α5β1 integrin, α_(v)β3 integrin, α_(v)β1 integrin, β1 integrin, 5T4,CAIX, CD4, CD13, CD19, CD20, CD22, CD25, CD30, CD31, CD33, CD34, CD40,CD44v6, CD45, CD51, CD52, CD54, CD56, CD64, CD70, CD74, CD79, CD105,CD117, CD123, CD133, CD138, CD144, CD146, CD152, CD174, CD205, CD227,CD326, CD340, Cripto, ED-B, GD2, TMEFF2, VEGFR1, VEGFR2, FGFR, PDGFR,ANGPT1, TIE1, TIE2, NRP1, TEK (CD202B), TGFβR, Death Receptor 5(Trail-R2), DLL4, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7,EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, FAP,GPNMB, ICAMs, VCAMs, PSMA, HER-2/neu, IL-13R alpha 2, MUC-1, MUC16,EGFR1 (HER-1), EGFR2 (HER-2/neu), EGFR3 (HER-3), IGF-1R, IGF-2R, c-Met(HGFR), Mesothelin, PDGFR, EDGR, TAG-72, transferrin receptor, EpCAM,CTLA-4, PSMA, tenascin C, alpha-fetoprotein, vimentin, C242 antigen,TRAIL-R1, TRAIL-R2, CA-125, GPNMB, CA-IX, GD3 ganglioside, RANKL, BAFF,IL-6R, TAG-72, HAMA, and CD166. Previously described target-specificantibodies that are used as the targeting component, in someembodiments, include but are not limited to mAb 3F8, mAb CSL362, mAb360, mAb J591, Abagovomab, Abciximab, Adalimumab, Afelimomab,Afutuzumab, Alacizumab, ALD518, Alemtuzumab, Altumomab, Anatumomab,Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atlizumab,Atorolimumab, Bapineuzmab, Basiliximab, Bavituximab, Bectumomab,Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab,Biciromab, Bivatuzumab, Blinatumomab, Brentuximab, Briakinumab,Canakinumab, Cantuzumab, Capromab, Catumaxomab, CC49, Cedelizumab,Certolizumab, Cetuximab, mAb528, Citatuzumab, Cixutumumab, Clenoliximab,Clivatuzumab, Conatumumab, CR6261, Dacetuzumab, Daclizumab, Daratumumab,Denosumab, Detumomab, Dorlimomab, Dorlixizumab, Ecromeximab, Eculizumab,Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab, Elsilimomab,Enlimomab, Epitumomab, Epratuzumab, Erlizumab, Ertumaxomab,Etaracizumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab,Felvizumab, Fezakinumab, Figitumumab, Fontolizumab, Foravirumab,Fresolimumab, Galiximab, Gantenerumab, Gavilimomab, Gemtuzumab,Girentuximab, Glembatumumab, Golimumab, Gomiliximab, Ibalizumab,Irbitumomab, Igovomab, Imciromab, Infliximab, Intetumumab, Inolimomab,Inotuzumab, Ipilimumab, Iratumumab, J591, Keliximab, Labetuzumab,Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab,Lintuzumab, Lorvotuzumab, Lucatumumab, Lumiliximab, Mapatumumab,Maslimomab, Matuzumab, Mepolizomab, Metelimumab, Milatuzumab,Minretumomab, Mitumomab, Morolimumab, Motavizumab, Muromonab, Nacolomab,Naptumomab, Natalizumab, Nebacumab, Necitutumab, Nerelimomab,Nimotuzumab, Nofetumomab, Ocrelizumab, Odulimomab, Ofatumumab,Olaratumab, Omalizumab, Oportuzumab, Oregovomab, Otelixizumab,Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Pascolizumab,Pemtumomab, Pertuzumab, Pexelizumab, Pintumomab, Priliximab, Pritumumab,PRO140, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab,Resilizumab, Rilotumumab, Rituximab, Robatumumab, Rontalizumab,Rovelizumab, Ruplizumab, Satumomab, Sevirumab, Sibrotuzumab,Sifalimumab, Siltuximab, Siplizumab, Solanezumab, Sonepcizumab,Sontuzumab, Stamulumab, Sulesomab, Tacatuzumab, Tadocizumab, Talizumab,Tanezumab, Taplitumomab, Tefibazumab, Telimomab, Tenatumomab,Teplizumab, TGN1412, Ticilimumab, Tigatuzumab, TNX-650, Tocilizumab,Toralizumab, Tositumomab, Trastuzumab, Tremelimumab, Tucotuzumab,Tuvirumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab,Veltuzumab, Vepalimomab, Visilizumab, Volociximab, Votumumab,Zalutumumab, Zanolimumab, Ziralimumab, Zolimomab, and any combination ofthe preceding.

4. Loading Payloads into Minicells

Eubacterial minicells are capable of encapsulating and deliveringseveral classes of biologically active compounds that have therapeutic,prophylactic, or diagnostic benefit to an animal. Types of thebiologically active compounds (payloads) that can be delivered byminicells include but are not limited to small molecules (includingsmall molecule drugs), nucleic acids, polypeptides, radioisotope,lipids, lipopolysaccharides, and any combination thereof.

Small molecules can include any number of therapeutic agents presentlyknown and used, or can be small molecules synthesized in a library ofsuch molecules for the purpose of screening for biological function(s).

Some embodiments relate to inducing the minicell production phenotypefrom an optimized eubacterial minicell-producing strain from, but notlimited to, the family Enterobacteriaceae such that it may be “loaded”with small molecule(s) including but not limited to a drug, a pro-drug,or a hormone incorporated following purification of the Fc-bindingminicells from the parental cells. Following production of the desiredquantity of “empty” Fc-binding minicells from a given culture andcondition, activation of the genetic suicide mechanism would beaccomplished by exposure of the culture or cells to a known signal.Following purification, Fc-binding minicells are “loaded” with the smallmolecule(s) by a simple incubation with a high concentration of thesmall molecule at a temperature ranging from 4° C. to 65° C. Furtherdetails regarding the loading of small molecules, including many ofthose listed herein, are known in the art.

Small molecules include without limitation organic compounds,peptidomimetics and fusion/conjugates thereof. As used herein, the term“organic compound” refers to any carbon-based compound other than themacromolecules nucleic acids and polypeptides. In addition to carbon,organic compounds can contain calcium, chlorine, fluorine, copper,hydrogen, iron, potassium, nitrogen, oxygen, sulfur and other elements.An organic compound may be in an aromatic or aliphatic form.Non-limiting examples of organic compounds include acetones, alcohols,anilines, carbohydrates, monosaccharides, oligosaccharides,polysaccharides, amino acids, nucleosides, nucleotides, lipids,retinoids, steroids, proteoglycans, ketones, aldehydes, saturated,unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters,ethers, thiols, sulfides, cyclic compounds, heterocylcic compounds,imidizoles, and phenols. An organic compound as used herein alsoincludes nitrated organic compounds and halogenated (e.g., chlorinated)organic compounds.

Small molecules can be synthetic, naturally occurring, and purified froma natural source. Examples of small molecules include, but are notlimited to, small molecule drugs, toxins, radionuclides, and smallmolecule imaging agents. Types of small molecule drugs include thosethat prevent, inhibit, stimulate, mimic, or modify a biological orbiochemical process within a cell, tissue type, or organ to the benefitof an animal suffering from a disease, whether somatic, germinal,infectious, or otherwise. Examples of drugs include chemotherapeuticagents (cancer drugs), antibiotics, antivirals, antidepressants,antihistamines, anticoagulants, and any other class or subclass thereofas listed in the Physicians' Desk Reference. Small molecules alsoinclude the class of molecules collectively known as fluorophores.Minicells encapsulating fluorophores and displaying cell-specifictargeting moieties can be used for in vivo imaging of cell types,tissues, organs, or tumors in an animal. Small molecule fluorophoresinclude but are not limited to DAPI, Cybr Gold, Cybr Green, EthidiumBromide, Alexa Flour, Texas Red, CFSE, and the like. Other types ofmolecular imaging agents are selected from the group including but notlimited to Gadolinium, ⁶⁴Cu diacetyl-bis(N⁴-methylthiosemicarbazone),¹⁸F-flourodeoxyglucose, ¹⁸F-flouride, 3′-deoxy-3′-[¹⁸F]fluorothymidine,¹⁸F-fluoromisonidazole, gallium, technetium-99, thallium, barium,gastrografin, iodine contrasting agents, iron oxide, green fluorescentprotein, luciferase, beta-galactosidase, and any combination of thepreceding.

Small molecule chemotherapeutic agents can be targeted and delivered totissues, cells, and organs using minicells displaying targetingmolecules. The term “chemotherapeutic agent” used herein refers toanti-cancer, anti-metastatic, anti-angiogenic, and otheranti-proliferative agents. In some embodiments, a chemotherapeutic agentis a chemical agent intended to inhibit the proliferation of or killcells. Examples of chemotherapeutic agent include, but are not limitedto: (1) DNA damaging agents and agents that inhibit DNA synthesis suchas anthracyclines (doxorubicin, daunorubicin, epirubicin), alkylatingagents (bendamustine, busulfan, carboplatin, carmustine, cisplatin,chlorambucil, cyclophosphamide, dacarbazine, hexamethylmelamine,ifosphamide, lomustine, mechlorethamine, melphalan, mitotane, mytomycin,pipobroman, procarbazine, streptozocin, thiotepa, andtriethylenemelamine), platinum derivatives (cisplatin, carboplatin, cisdiamminedichloroplatinum), telomerase and topoisomerase inhibitors(Camptosar), (2) microtubule and tubulin binding agents including butnot limited to taxanes and taxane derivatives (paclitaxel, docetaxel,BAY 59-8862), (3) anti-metabolites such as capecitabine,chlorodeoxyadenosine, cytarabine (and its activated form, ara-CMP),cytosine arabinoside, dacarbazine, floxuridine, fludarabine,5-fluorouracil, 5-DFUR, gemcitabine, hydroxyurea, 6-mercaptopurine,methotrexate, pentostatin, trimetrexate, and 6-thioguanine (4)anti-angiogenics (thalidomide, sunitinib, lenalidomide), vasculardisrupting agents (flavonoids/flavones, DMXAA, combretastatinderivatives such as CA4DP, ZD6126, AVE8062A, etc.), (5) endocrinetherapy such as aromatase inhibitors (4-hydroandrostendione, exemestane,aminoglutethimide, anastrozole, letrozole), (6) anti-estrogens(Tamoxifen, Toremifene, Raloxifene, Faslodex), steroids such asdexamethasone, (7) immuno-modulators such as Toll-like receptor agonistsor antagonists, (8) inhibitors to integrins, other adhesion proteins andmatrix metalloproteinases), (9) histone deacetylase inhibitors, (10)inhibitors of signal transduction such as inhibitors of tyrosine kinaseslike imatinib (Gleevec), (11) inhibitors of heat shock proteins, (12)retinoids such as all trans retinoic acid, (13) inhibitors of growthfactor receptors or the growth factors themselves, (14) anti-mitoticcompounds such as navelbine, vinblastine, vincristine, vindesine, andvinorelbine, (15) anti-inflammatories such as COX inhibitors and (16)cell cycle regulators such as check point regulators and telomeraseinhibitors, (17) transcription factor inhibitors, and apoptosisinducers, such as inhibitors of Bcl-2, Bcl-x and XIAP.

Nucleic acids include DNA and RNA and their structural equivalents suchas RNA molecules or DNA molecules that utilize phosphorothioatebackbones as opposed to the naturally occurring phosphodiesterbackbones. DNA molecules include episomal DNA (not located on or part ofthe host cell chromosome) which further include plasmid DNA, cosmid DNA,bacteriophage DNA, and bacterial artificial chromosomes (BACs), and thelike. DNA molecules encode for proteins as described by the centraldogma of molecular biology. Thus DNA may encode for proteins of anyorigin, naturally occurring or synthetic. Likewise, DNA can beengineered to contain “promoter sequences” that are recognized by hostcell machinery to activate expression of the encoded proteins. Promotersequences can be cell specific, tissue specific, or inducer specific.Inducers are exogenously applied signals that help to activate thepromoters to produce the proteins. Inducers can be chemical or physicalin nature. Many promoter systems are known to those skilled in the artas are the sequences that render them functional. Preferred prokaryoticexpression sequences include but are not limited to the pRHA system, thepBAD system, the T7 polymerase system, the pLac system and its myriadderivatives, the pTet system, and the CI857ts system. Preferredeukaryotic promoter systems include but are not limited to the CMVpromoter, the SV40 promoter system, and the BGH promoter system.Examples of RNAs include, but are not limited to, messenger RNA (mRNA),transfer RNA (tRNA), and small nuclear RNAs. Many RNAs, classified asantisense RNAs, include but are not limited to small-interfering RNAs(siRNA), short hairpin RNAs (shRNAs), and full length antisense RNAs.Micro RNAs are also included. Preferred targets of siRNA or shRNAinclude but are not limited to Androgen Receptor (AR), ABCB1/MDR1/PGY1(P-glycoprotein; Pgp), CHK-1, HIF-1, Mcl-1, PDGFR, Tie-2, ABL1, ABL2,AKT2, ALK, BCL2, BCL3, BCL5, BCL6, BLC7A, BCL9, BCL10, BCL11A, BCL11B,Bcl-x, Bcr-Abl, BRAF, CCND1, CDK4, CHK-1, c-Met, c-myc, CTNNB1, DKC1,EGFR1, EGFR2, ERBB2, ERCC-1, EZH2, FES, FGFR1, FGFR2, FGFR3, FGFR-4,FLT1 (VEGFR1), FLT2, FLT3, FLT4, HER2, HER3, HRAS, IGFR, Interleukin 8(IL-8), JAK, JAK2, KDR/Flk-1 (VEGFR-2), KIT, KRAS2, MET, MRP, mTOR, MYC,MYCL1, MYCN, NRAS, p53, PARP1, PDGFB, PDGFRA, PDGFRB, PI3KCA, PPAR,Rad51, Rad52, Rad53, RalA, REL, RET, RRM1, RRM2, STATS, survivin,telomerase, TEP1, TERC, TERT, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E,Wnt-1, XIAP, and any against any nucleotide sequence from the list ofgenes above that contains somatic or germline mutations compared to thewild type gene sequence.

Proteins are comprised of polypeptides and are encoded by DNA. Proteinscan be biologically functional, such as enzymes, toxins, or signalingproteins. Proteins can be structural, such as is the case for actin andthe like. Proteins can provide localization signals by being fluorescentor bioluminescent. Proteins can serve as immunogens or serve othertherapeutic purposes (such as supplying or restoring enzyme in a targetcell, tissue, organ, or animal). Proteins can aid in thepost-endocytosis intracellular transfer of other payload types. Forexample, proteins such as listeriolysin O from Listeria monocytogenescan be employed to facilitate the transfer of the minicell payload(s)from the endocytotic compartment(s) of a target cell to the cytosol of atarget cell. Proteins can also be pro-drug converting enzymes. Preferredproteins include listeriolysin O, green fluorescent protein, redfluorescent protein and any member of the luciferase family of proteins.Recombinantly expressed/produced therapeutic polypeptides to bedelivered by targeted minicells include but are not limited to proteintoxins, cholesterol-dependent cytolysins, functional enzymes, activatedcaspases, pro-caspases, cytokines, chemokines, cell-penetratingpeptides, and any combination of the proceeding. Recombinant expressionof a therapeutic polypeptide(s) can be the result of expression from anyof the various episomal recombinant prokaryotic expression vectors knownin the art including but not limited to plasmids, cosmids, phagemids,and bacterial artificial chromosomes (BACs), and any combination of thepreceding. In similar fashion, recombinant expression can be achieved bya chromosomally located prokaryotic expression cassette present in oneor more copies of the minicell-producing parent cell chromosome. Thedelivery of protein toxins using the targeted minicells disclosed hereinis a particularly attractive approach in applications where selectiveelimination of cells in vivo is desirable. Protein toxins include butare not limited to gelonin, diphtheria toxin fragment A, diphtheriatoxin fragment A/B, tetanus toxin, E. coli heat labile toxin (LTI and/orLTII), cholera toxin, C. perfringes iota toxin, Pseudomonas exotoxin A,shiga toxin, anthrax toxin, MTX (B. sphaericus mosquilicidal toxin),perfringolysin O, streptolysin, barley toxin, mellitin, anthrax toxinsLF and EF, adenylate cyclase toxin, botulinolysin B, botulinolysin E3,botulinolysin C, botulinum toxin A, cholera toxin, clostridium toxins A,B, and alpha, ricin, shiga A toxin, shiga-like A toxin, cholera A toxin,pertussis S1 toxin, E. coli heat labile toxin (LTB), pH stable variantsof listeriolysin O (pH-independent; amino acid substitution L461T),thermostable variants of listeriolysin O (amino acid substitutionsE247M, D320K), pH and thermostable variants of listeriolysin O (aminoacid substitutions E247M, D320K, and L461T), streptolysin O,streptolysin O c, streptolysin O e, sphaericolysin, anthrolysin O,cereolysin, thuringiensilysin O, weihenstephanensilysin, alveolysin,brevilysin, butyriculysin, tetanolysin O, novyilysin, lectinolysin,pneumolysin, mitilysin, pseudopneumolysin, suilysin, intermedilysin,ivanolysin, seeligeriolysin O, vaginolysin, and pyolysin. Therapeuticpolypeptides may be localized to different sub-cellular compartments ofthe minicell at the discretion of the artisan. When targeted minicellsdisclosed herein are derived from a Gram-negative parentalminicell-producing strain, recombinantly expressed therapeuticpolypeptides produced therefrom can be localized to the cytosol, theinner leaflet of the inner membrane, the outer leaflet of the innermembrane, the periplasm, the inner leaflet of the outer membrane, theouter membrane of minicells, and any combination of the proceeding. Whentargeted minicells disclosed herein are derived from a Gram-positiveparental minicell-producing strain, recombinantly expressed therapeuticpolypeptides produced therefrom can be localized to the cytosol, thecell wall, the inner leaflet of the membrane, the membrane of minicells,and any combination of the proceeding.

Any and all of the payload types described herein can be used incombination or singular at the discretion of the user. One skilled inthe art will appreciate and recognize which combinations are to be usedfor which therapeutic purpose(s) (e.g., the combination of a smallmolecule cytotoxic cancer drug and an si/shRNA against a gene productconferring resistance to the drug).

5. Pharmaceutical Compositions

The present application also relates to compositions, including but notlimited to pharmaceutical compositions. The term “composition” usedherein refers to a mixture comprising at least one carrier, preferably aphysiologically acceptable carrier, and one or more minicellcompositions. The term “carrier” used herein refers to a chemicalcompound that does not inhibit or prevent the incorporation of thebiologically active peptide(s) into cells or tissues. A carriertypically is an inert substance that allows an active ingredient to beformulated or compounded into a suitable dosage form (e.g., a pill, acapsule, a gel, a film, a tablet, a microparticle (e.g., a microsphere),a solution; an ointment; a paste, an aerosol, a droplet, a colloid or anemulsion etc.). A “physiologically acceptable carrier” is a carriersuitable for use under physiological conditions that does not abrogate(reduce, inhibit, or prevent) the biological activity and properties ofthe compound. For example, dimethyl sulfoxide (DMSO) is a carrier as itfacilitates the uptake of many organic compounds into the cells ortissues of an organism. Preferably, the carrier is a physiologicallyacceptable carrier, preferably a pharmaceutically or veterinarilyacceptable carrier, in which the minicell composition is disposed.

A “pharmaceutical composition” refers to a composition wherein thecarrier is a pharmaceutically acceptable carrier, while a “veterinarycomposition” is one wherein the carrier is a veterinarily acceptablecarrier. The term “pharmaceutically acceptable carrier” or “veterinarilyacceptable carrier” used herein includes any medium or material that isnot biologically or otherwise undesirable, i.e., the carrier may beadministered to an organism along with a minicell composition withoutcausing any undesirable biological effects or interacting in adeleterious manner with the complex or any of its components or theorganism. Examples of pharmaceutically acceptable reagents are providedin The United States Pharmacopeia, The National Formulary, United StatesPharmacopeial Convention, Inc., Rockville, Md. 1990, hereby incorporatedby reference herein into the present application. The terms“therapeutically effective amount” and “pharmaceutically effectiveamount” refer to an amount sufficient to induce or effectuate ameasurable response in the target cell, tissue, or body of an organism.What constitutes a therapeutically effective amount will depend on avariety of factors, which the knowledgeable practitioner will take intoaccount in arriving at the desired dosage regimen.

The compositions can also comprise other chemical components, such asdiluents and excipients. A “diluent” is a chemical compound diluted in asolvent, preferably an aqueous solvent, that facilitates dissolution ofthe composition in the solvent, and it may also serve to stabilize thebiologically active form of the composition or one or more of itscomponents. Salts dissolved in buffered solutions are utilized asdiluents in the art. For example, preferred diluents are bufferedsolutions containing one or more different salts. An unlimiting exampleof preferred buffered solution is phosphate buffered saline(particularly in conjunction with compositions intended forpharmaceutical administration), as it mimics the salt conditions ofhuman blood. Since buffer salts can control the pH of a solution at lowconcentrations, a buffered diluent rarely modifies the biologicalactivity of a given compound or pharmaceutical composition.

An “excipient” is any more or less inert substance that can be added toa composition in order to confer a suitable property, for example, asuitable consistency or to produce a drug formulation. Suitableexcipients and carriers include, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol cellulose preparationssuch as, for example, maize starch, wheat starch, rice starch, agar,pectin, xanthan gum, guar gum, locust bean gum, hyaluronic acid, caseinpotato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, polyacrylate, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents can also be included, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. Other suitable excipients and carriers includehydrogels, gellable hydrocolloids, and chitosan. Chitosan microspheresand microcapsules can be used as carriers. See e.g., WO 98/52547 (whichdescribes microsphere formulations for targeting compounds to thestomach, the formulations comprising an inner core (optionally includinga gelled hydrocolloid) containing one or more active ingredients, amembrane comprised of a water insoluble polymer (e.g., ethylcellulose)to control the release rate of the active ingredient(s), and an outerlayer comprised of a bioadhesive cationic polymer, for example, acationic polysaccharide, a cationic protein, and/or a synthetic cationicpolymer; U.S. Pat. No. 4,895,724. Typically, chitosan is cross-linkedusing a suitable agent, for example, glutaraldehyde, glyoxal,epichlorohydrin, and succinaldehyde. Compositions employing chitosan asa carrier can be formulated into a variety of dosage forms, includingpills, tablets, microparticles, and microspheres, including thoseproviding for controlled release of the active ingredient(s). Othersuitable bioadhesive cationic polymers include acidic gelatin,polygalactosamine, polyamino acids such as polylysine, polyhistidine,polyornithine, polyquaternary compounds, prolamine, polyimine,diethylaminoethyldextran (DEAE), DEAE-imine, DEAE-methacrylate,DEAE-acrylamide, DEAE-dextran, DEAE-cellulose, poly-p-aminostyrene,polyoxethane, copolymethacrylates, polyamidoamines, cationic starches,polyvinylpyridine, and polythiodiethylaminomethylethylene.

The compositions can be formulated in any suitable manner. Minicellcompositions may be uniformly (homogeneously) or non-uniformly(heterogeneously) dispersed in the carrier. Suitable formulationsinclude dry and liquid formulations. Dry formulations include freezedried and lyophilized powders, which are particularly well suited foraerosol delivery to the sinuses or lung, or for long term storagefollowed by reconstitution in a suitable diluent prior toadministration. Other preferred dry formulations include those wherein acomposition disclosed herein is compressed into tablet or pill formsuitable for oral administration or compounded into a sustained releaseformulation. When the composition is intended for oral administration tobe delivered to epithelium in the intestines, it is preferred that theformulation be encapsulated with an enteric coating to protect theformulation and prevent premature release of the minicell compositionsincluded therein. As those in the art will appreciate, the compositionsdisclosed herein can be placed into any suitable dosage form. Pills andtablets represent some of such dosage forms. The compositions can alsobe encapsulated into any suitable capsule or other coating material, forexample, by compression, dipping, pan coating, spray drying, etc.Suitable capsules include those made from gelatin and starch. In turn,such capsules can be coated with one or more additional materials, forexample, and enteric coating, if desired. Liquid formulations includeaqueous formulations, gels, and emulsions.

Some preferred embodiments provide compositions that comprise abioadhesive, preferably a mucoadhesive, coating. A “bioadhesive coating”is a coating that allows a substance (e.g., a minicell composition) toadhere to a biological surface or substance better than occurs absentthe coating. A “mucoadhesive coating” is a preferred bioadhesive coatingthat allows a substance, for example, a composition to adhere better tomucosa occurs absent the coating. For example, minicells can be coatedwith a mucoadhesive. The coated particles can then be assembled into adosage form suitable for delivery to an organism. Preferably, anddepending upon the location where the cell surface transport moiety tobe targeted is expressed, the dosage form is then coated with anothercoating to protect the formulation until it reaches the desiredlocation, where the mucoadhesive enables the formulation to be retainedwhile the composition interacts with the target cell surface transportmoiety.

Compositions disclosed herein can be administered to any organism,preferably an animal, preferably a mammal, bird, fish, insect, orarachnid. Preferred mammals include bovine, canine, equine, feline,ovine, and porcine animals, and non-human primates. Humans areparticularly preferred. Multiple techniques of administering ordelivering a compound exist in the art including, but not limited to,oral, rectal (e.g. an enema or suppository) aerosol (e.g., for nasal orpulmonary delivery), parenteral, and topical administration. Preferably,sufficient quantities of the biologically active peptide are deliveredto achieve the intended effect. The particular amount of composition tobe delivered will depend on many factors, including the effect to beachieved, the type of organism to which the composition is delivered,delivery route, dosage regimen, and the age, health, and sex of theorganism. As such, the particular dosage of a composition incorporatedinto a given formulation is left to the ordinarily skilled artisan'sdiscretion.

Those skilled in the art will appreciate that when the compositionsdisclosed herein are administered as agents to achieve a particulardesired biological result, which may include a therapeutic, diagnostic,or protective effect(s) (including vaccination), it may be possible tocombine the minicell composition with a suitable pharmaceutical carrier.The choice of pharmaceutical carrier and the preparation of theminicells as a therapeutic or protective agent will depend on theintended use and mode of administration. Suitable formulations andmethods of administration of therapeutic agents include those for oral,pulmonary, nasal, buccal, ocular, dermal, rectal, intravenous, orvaginal delivery.

Depending on the mode of delivery employed, the context-dependentfunctional entity can be delivered in a variety of pharmaceuticallyacceptable forms. For example, the context-dependent functional entitycan be delivered in the form of a solid, solution, emulsion, dispersion,and the like, incorporated into a pill, capsule, tablet, suppository,aerosol, droplet, or spray. Pills, tablets, suppositories, aerosols,powders, droplets, and sprays may have complex, multilayer structuresand have a large range of sizes. Aerosols, powders, droplets, and spraysmay range from small (1 micron) to large (200 micron) in size.

Pharmaceutical compositions disclosed herein can be used in the form ofa solid, a lyophilized powder, a solution, an emulsion, a dispersion,and the like, wherein the resulting composition contains one or more ofthe compounds disclosed herein, as an active ingredient, in admixturewith an organic or inorganic carrier or excipient suitable for enteralor parenteral applications. The active ingredient may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, solutions, emulsions,suspensions, and any other form suitable for use. The carriers which canbe used include glucose, lactose, mannose, gum acacia, gelatin,mannitol, starch paste, magnesium trisilicate, talc, corn starch,keratin, colloidal silica, potato starch, urea, medium chain lengthtriglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition auxiliary, stabilizing, thickening and coloring agents andperfumes may be used. Examples of a stabilizing dry agent includetriulose, preferably at concentrations of 0.1% or greater (See, e.g.,U.S. Pat. No. 5,314,695). The active compound is included in thepharmaceutical composition in an amount sufficient to produce thedesired effect upon the process or condition of diseases.

6. Therapeutic Indications

The present application relates to diagnostic imaging and therapy ofcancer(s) including but not limited to solid tumors, metastatic tumors,and liquid tumors. Solid and metastatic tumors include those ofepithelial origin and include but are not limited to breast, lung,pancreatic, prostatic, testicular, ovarian, gastric, intestinal, mouth,tongue, pharynx, hepatic, anal, rectal, colonic, esophageal, urinarybladder, gall bladder, skin, uterine, vaginal, penal, and renal cancers.Other solid cancer types that may be treated with the targeted minicellsdisclosed herein include but are not limited to adenocarcinomas,sarcomas, fibrosarcomas, and cancers of the eye, brain, and bone. Liquidtumors that can be treated by the targeted minicells disclosed hereininclude but are not limited to non-Hodgkin's lymphoma, myeloma,Hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocyticleukemia, acute myeloid leukemia, chronic myeloid leukemia, and otherleukemias. The targeted minicells disclosed herein are targeted toeukaryotic cancer cell-specific surface antigens that include but arenot limited to α3β1 integrin, α4β1 integrin, α5β1 integrin,α_(v)β3integrin, α_(v)β1integrin, β1 integrin, 5T4, CAIX, CD4, CD13,CD19, CD20, CD22, CD25, CD30, CD31, CD33, CD34, CD40, CD44v6, CD45,CD51, CD52, CD54, CD56, CD64, CD70, CD74, CD79, CD105, CD117, CD123,CD133, CD138, CD144, CD146, CD152, CD174, CD205, CD227, CD326, CD340,Cripto, ED-B, GD2, TMEFF2, VEGFR1, VEGFR2, FGFR, PDGFR, ANGPT1, TIE1,TIE2, NRP1, TEK (CD202B), TGFβR, Death Receptor 5 (Trail-R2), DLL4,EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10,EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, FAP, GPNMB, ICAMs, VCAMs,PSMA, HER-2/neu, IL-13R alpha 2, MUC-1, MUC16, EGFR1 (HER-1), EGFR2(HER-2/neu), EGFR3 (HER-3), IGF-1R, IGF-2R, c-Met (HGFR), Mesothelin,PDGFR, EDGR, TAG-72, transferrin receptor, EpCAM, CTLA-4, PSMA, tenascinC, alpha-fetoprotein, vimentin, C242 antigen, TRAIL-R1, TRAIL-R2,CA-125, GPNMB, CA-IX, GD3 ganglioside, RANKL, BAFF, IL-6R, TAG-72, HAMA,and CD166.

The present application also relates to diagnostic imaging and therapyof conditions and diseases in an animal caused, at least in part, byaberrant vasculogenesis or angiogenesis. Such conditions and diseasesinclude but are not limited to cancer, inflammatory conditions,including, but not limited to, rheumatoid arthritis, psoriasis andinflammatory bowel disease, metabolic disorders, including diabeticretinopathy and diabetic nephropathy and ocular conditions, including,but not limited to, neovascular (wet) AMD and macular edema. A role forvasculogenesis or angiogenesis has been established in each of thesediseases or conditions as a result of genetic, mechanistic,histopathological, preclinical and/or clinical studies. For example,tumors cannot grow beyond 1 to 2 mm in diameter in the absence ofneovascularization. The important role of neovascularization in cancerand certain ocular diseases has been validated clinically via theapproval of several anti-angiogenic therapeutics, including bevacizumabfor cancer and ranibizumab for AMD. In addition, aberrant vascularremodeling and angiogenesis play an important role in several stages ofinflammation. The first acute phase of inflammation involves functionalchanges in vasculature, such as dilation, increased permeability andendothelial cell activation. The second subacute phase of inflammationinvolves capillary and venule remodeling, with extensive endothelialmitotic activity. In the chronic setting, neovascularization and/orexpansion of microvasculature is observed, including in rheumatoidarthritis, psoriasis, diabetic retinopathy and diabetic nephropathy. Allof these vascular changes promote and sustain inflammatory responses byenhancing infiltration and/or release of nutrients, cytokines,chemokines, proteases and inflammatory leukocytes. Thus, targetedminicells described herein can be used as anti-angiogenic therapeuticsby incorporating antibodies that recognize cell surface antigens of celltypes contributing to the misregulation of angiogenesis in a givendisease setting. By way of non-limiting example, endothelial cells,circulating endothelial cells, angioblasts, hemangioblasts, pericytes,myofibroblasts, and endothelial progenitor cells are all targets for theprevention of vasculogenesis or angiogenesis. Endothelial cells andtheir progenitors, in particular, are critical vasculogenic andangiogenic cell types that can be targeted using anti-angiogenicminicells. Many endothelial cells overexpress, preferentially express,and/or differentially express distinct cell surface proteins at sites ofvasculogenesis and angiogenesis. Additionally, circulating endothelialcells and circulating endothelial progenitor cells (present in the bloodand lymph) are targets for anti-angiogenic minicells. Circulatingendothelial cells and endothelial progenitors also express cell surfaceantigens that distinguish them from other cell types, serving as a basisfor the preferential targeting of anti-angiogenic minicells.Collectively, these cell surface molecules are termedangiogenesis-specific antigens. Many antibodies that specificallyrecognize angiogenesis-specific antigens, and nucleic acid sequences ofthe variable regions thereof, are already known in the art. Any of theseantibodies can be used in exogenous fashion with the presentapplication. Many angiogenesis-specific antigens have been identified towhich no reported antibodies exist. However, methods to produceantibodies to these antigens are well known in the art and any and allantibodies to angiogenesis-specific antigens, or any otherangiogenesis-related surface antigen, can be incorporated into thecomposition as described. Angiogenesis-specific antigens of choiceinclude, but are not limited to α4β1 integrin, α5β1 integrin,α_(v)β3integrin, α_(v)β1integrin, β1 integrin, CD13, CD31, CD34, CD45,CD54, CD105, CD117, CD133, CD144, CD146, VEGFR1, VEGFR2, FGFR, PDGFR,ANGPT1, TIE1, TIE2, NRP1, TEK (CD202B), TGFβR, DLL4, EPHA1, EPHA2,EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10 EPHB1, EPHB2,EPHB3, EPHB4, EPHB5, EPHB6, ICAMs, VCAMs, and PSMA.

7. Minicell Preparations

Some embodiments relate to creating an optimized strain and preparingFc-binding minicells from, but not limited to, the familyEnterobacteriaceae that contains or produces any subclass of therapeuticRNA, including but not limited to antisense RNA (siRNA and shRNA as anexample), ribozymes, and miRNA such that the resulting minicellscomprise an enriched amount of the therapeutic RNA molecules by way ofencapsulation after expression of the therapeutic RNA molecule by theparental cell or the minicells themselves. Following production of thedesired quantity of minicells from the culture and condition, activationof the genetic suicide mechanism would be accomplished by exposure ofthe culture or cells to a known signal. Alternatively, loading of any ofthe above RNA molecules into Fc-binding minicells can also beaccomplished by incubating minicells with high concentrations ofexogenous RNA molecules (as opposed to, or in combination with,expression of the same or different therapeutic RNA by theminicell-producing parental strain such that the resulting minicellscomprise the therapeutic RNA).

Some embodiments relate to creating an optimized strain and preparingFc-binding minicells from, but not limited to, the familyEnterobacteriaceae that contain or produce a protein molecule, such thatthe resulting minicells contain the protein molecule by way ofencapsulation after expression of the protein molecule by the parentalcell or by the minicells themselves. Following production of the desiredquantity of minicells from a given culture and condition, activation ofthe genetic suicide mechanism would be accomplished by exposure of theculture or cells to a known signal.

Some embodiments relate to creating an optimized strain and preparingFc-binding minicells from, but not limited to, the familyEnterobacteriaceae that contains or produces DNA molecules (e.g. aeukaryotic expression plasmid) encoding for a therapeutic or deleteriousgene or gene product, any subclass of RNA, and/or proteins, such thatthe resulting minicells contain the combination of molecules by way ofencapsulation. Following production of the desired quantity of minicellsfrom a given culture and condition, activation of the genetic suicidemechanism would be accomplished by exposure of the culture or cells to aknown signal.

Some embodiments relate to creating an optimized strain and preparingFc-binding minicells from, but not limited to, the familyEnterobacteriaceae such that the minicells may be “loaded” with smallmolecules that comprise but are not limited to a drug, a pro-drug, or ahormone following purification. Following production of the desiredquantity of minicells from a given culture and condition, activation ofthe genetic suicide mechanism would be accomplished by exposure of theculture or cells to a known signal. Following purification, minicellswould be “loaded” with the small molecule(s) by incubation with a highconcentration of the small molecule at a temperature ranging from 0° C.to 65° C. This procedure is performed with minicells with “empty”Fc-binding minicells such that the small molecule is the only exogenoustherapeutic molecule in the resulting targeted minicells. This proceduremay also be performed with minicells that further comprise anycombination of therapeutic DNA, therapeutic RNA, or therapeutic protein,such that the end composition contains the small molecule and anycombination of the therapeutic DNA, therapeutic RNA, and/or therapeuticprotein. The Fc-binding minicells are the made targeting competent bythe addition of antibodies and/or Fc-fusion/conjugated targetingmolecules on their surfaces.

Some embodiments relate to creating an optimized strain and preparingFc-binding minicells from, but not limited to, the family Bacillaceaethat contains or produces a DNA molecule encoding for a therapeutic ordeleterious gene or gene product, such that the resulting minicellcontains the DNA molecule by way of encapsulation. Following productionof the desired quantity of minicells from a given culture and condition,activation of the genetic suicide mechanism would be accomplished byexposure of the culture or cells to a known signal.

Some embodiments relate to creating an optimized strain and preparingFc-binding minicells from, but not limited to, the family Bacillaceaethat contains or produces any subclass of therapeutic RNA, including butnot limited to antisense RNA (siRNA and shRNA as an example), ribozymes,and miRNA such that the resulting minicells comprise an enriched amountof the therapeutic RNA molecules by way of encapsulation afterexpression of the therapeutic RNA molecule by the parental cell or theminicells themselves. Following production of the desired quantity ofminicells from the culture and condition, activation of the geneticsuicide mechanism would be accomplished by exposure of the culture orcells to a known signal. Alternatively, loading of any of the above RNAmolecules into Fc-binding minicells can also be accomplished byincubating minicells with high concentrations of exogenous RNA molecules(as opposed to, or in combination with, expression of the same ordifferent therapeutic RNA by the minicell-producing parental strain suchthat the resulting minicells comprise the therapeutic RNA).

Some embodiments relate to creating an optimized strain and preparingFc-binding minicells from, but not limited to, the family Bacillaceaethat contains or produces a protein molecule, such that the resultingminicell contains the protein molecule by way of encapsulation afterexpression of the protein molecule by the parental cell or by theminicell itself. Following production of the desired quantity ofminicells from a given culture and condition, activation of the geneticsuicide mechanism would be accomplished by exposure of the culture orcells to a known signal.

Some embodiments relate to creating an optimized strain and preparingFc-binding minicells from, but not limited to, the family Bacillaceaethat contains or produces a predetermined and deliberate combination ofDNA molecules encoding for a therapeutic or deleterious gene or geneproduct, any subclass of RNA, and/or proteins, such that the resultingminicell contains the combination of molecules by way of encapsulation.Following production of the desired quantity of minicells from a givenculture and condition, activation of the genetic suicide mechanism wouldbe accomplished by exposure of the culture or cells to a known signal.The signal would be applied in each step of the purification process toensure maximal killing of viable cells in the final preparation.

Some embodiments relate to creating an optimized strain and preparingFc-binding minicells from, but not limited to, the family Bacillaceaesuch that the minicells may be “loaded” with small molecules thatcomprise but are not limited to a drug, a pro-drug, or a hormonefollowing purification. Following production of the desired quantity ofminicells from a given culture and condition, activation of the geneticsuicide mechanism would be accomplished by exposure of the culture orcells to a known signal. Following purification, minicells would be“loaded” with the small molecule(s) by incubation with a highconcentration of the small molecule at a temperature ranging from 0 to65° C. This procedure is performed with minicells with “empty”Fc-binding minicells such that the small molecule is the only exogenoustherapeutic molecule species in the resulting targeted minicells. Thisprocedure may also be performed with minicells that further comprise anycombination of therapeutic DNA, therapeutic RNA, or therapeutic protein,such that the end composition contains the small molecule and anycombination of the therapeutic DNA, therapeutic RNA, and/or therapeuticprotein. The Fc-binding minicells are the made targeting competent bythe addition of antibodies and/or Fc-fusion/conjugated targetingmolecules on their surfaces.

In some embodiments, the level of minicell producing parental cellcontamination is less than 1 in 10⁷ targeted therapeutic minicells.

In some embodiments, the level of minicell producing parental cellcontamination is less than 1 in 10⁸ targeted therapeutic minicells.

In some embodiments, the level of minicell producing parental cellcontamination is less than 1 in 10⁹ targeted therapeutic minicells.

In some embodiments, the level of minicell producing parental cellcontamination is less than 1 in 10¹⁰ targeted therapeutic minicells.

In some embodiments, the level of minicell producing parental cellcontamination is less than 1 in 10¹¹ targeted therapeutic minicells.

In some embodiments, the level of minicell producing parental cellcontamination is less than 1 in 10¹² targeted therapeutic minicells.

In some embodiments, the level of minicell producing parental cellcontamination is less than 1 in 10¹³ targeted therapeutic minicells.

In some embodiments, the level of minicell producing parental cellcontamination is less than 1 in 10¹⁴ targeted therapeutic minicells.

In some embodiments, the level of minicell producing parental cellcontamination is less than 1 in 10¹⁵ targeted therapeutic minicells.

In some embodiments, the level of minicell producing parental cellcontamination is less than 1 in 10¹⁶ targeted therapeutic minicells.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although the present application has been described withreference to embodiments and examples, it should be understood thatvarious modifications can be made without departing from the spirit ofthe invention. All references cited herein are expressly incorporatedherein by reference in their entirety.

Embodiments of the present application are disclosed in further detailin the following examples, which are not in any way intended to limitthe scope of the present application.

EXAMPLES Example 1

Expression and display of the Fc binding region of Protein A or ProteinG on the surface of minicells demonstrated by enzyme linkedimmunoabsorbent assay (ELISA). Minicell-producing E. coli strain VAX12B4was transformed with either (i) the L-rhamnose inducible expressionplasmid pVX-119 (codes for Lpp-OmpAΩProtein A; SEQ ID NO:1), (ii) theL-rhamnose inducible expression plasmid pVX-120 (codes forLpp-OmpAΩProtein G; SEQ ID NO:2) or (iii) with an empty vector controlplasmid, to create minicell-producing strains VAX13B7 (Protein A),VAX13C4 (Protein G), and VAX12C4 (vector control), respectively. Each ofVAX13B7, VAX13C4, and VAX12C4 were grown overnight in 20 mL ofLuria-Bertani (LB) broth containing 0.2% glucose, 10 mg/mLdiaminopimelic acid (DAP), 11 mg/mL lysine and 50 mg/mL Kanamycin. Thefollowing day, each strain was independently subcultured by a 1/100dilution of the overnight culture into 700 mL of fresh LB brothcontaining DAP, lysine and kanamycin, as above. Cultures were grown toan optical density (O.D.₆₀₀) of 0.1 at which time L-rhamnose was addedto a final concentration of 10 micromolar to induce fusion proteinexpression. When the cultures reached an O.D.₆₀₀ of 1.0, 20 micromolarIsopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induceminicell formation. Eight hours post L-rhamnose induction, the cultureswere transferred to 42° C. and incubated overnight to induce parentalcell suicide. Minicells were then purified from the culture by sucrosedensity fractionation, and analyzed for surface display of eitherProtein A or Protein G by ELISA. ELISAs were performed by incubating1e07 minicells derived from VAX13B7, VAX13C4, or VAX12C4 in sodiumbicarbonate buffer (pH 9.5) in a 96-well polystyrene plate overnight toallow minicells to bind to the plate. The following day, plate wellswere washed three times each with phosphate buffered saline, pH 7.4(PBS) containing 0.05% Tween-20, and then blocked using PBS containing1% gelatin for 1 hour at room temperature. Wells were then washed threetimes each with PBS containing 0.05% Tween-20 and then incubated with anHRP-conjugated chicken IgY antibody against either Protein A or ProteinG for 1 hour at room temperature. Following incubation, the plates werewashed five times each with PBS containing 0.5% Tween-20, and then TMBwas added to each well. Reactions were stopped before the standard(recombinant Protein A/G) signal was saturated, by the addition of 1Msulfuric acid and the plates were then analyzed on a SpectraMax M3 platereader (Molecular Devices, Inc.). The level of surface fusion proteindisplay was determined by comparing the experimental ELISA signal to astandard curve created by titration of recombinant Protein A/G (Pierce,Inc.) and shown in FIG. 2.

As shown in FIG. 2, protein A-displaying minicells were only detectedwhen an HRP-conjugated anti-Protein A chicken IgY secondary antibody wasused. Protein G-displaying minicells were only detected when anHRP-conjugated anti-Protein G chicken IgY secondary antibody was used.These results confirmed the expression, identity and minicell surfacedisplay of the fusion proteins.

Example 2

Binding and display of VEGFR2 antibody by miniciells expressing the Fcbinding portion of Protein A or Protein G. Minicells (1e09) purifiedfrom strain VAX13B7 (Protein A-displaying), VAX13C4 (ProteinG-displaying), and VAX12B5 (negative control) were incubated with 1microgram each without (−) or with (+) a mouse monoclonal IgG antibodyagainst human VEGFR2 for 1 hour at room temperature to allow antibodiesto bind to the minicells. After incubation, minicells were washed threetimes each with 1 mL of PBS (pH 7.4) to remove any unbound antibody.Minicells (1e08) were then analyzed by Western blot using anHRP-conjugated rabbit anti-mouse polyclonal antibody as the secondaryantibody. The Western Blot is shown in FIG. 3. Specific binding of thesecondary antibody was visualized using an Amersham ECL Detection Kit(GE Healthcare). Mouse anti-VEGFR2 antibody (100 ng) was loaded as apositive control (lane after 12B5).

FIG. 3 shows that the Fc region of the HRP-conjugated rabbit anti-mousesecondary antibody was bound by the Protein A and Protein G fusionproteins in the 13B7 and 13C4 minicells (49.1 kDa and 38.5 kDa,respectively), independent of the VEGFR2 antibody. In addition, the Fabregion of the secondary antibody binds to and detects the intact VEGFR2antibody (˜150 kDa) bound to the 13B7 and 13C4 minicells.

Example 3

Binding and display of EGFR1 antibody by miniciells expressing the Fcbinding portion of Protein A or Protein G. Minicells (1e09) purifiedfrom strain VAX13B7 (Protein A-displaying), VAX13C4 (ProteinG-displaying), and VAX12B5 (negative control) were incubated with 1microgram each without (−) or with (+) a mouse monoclonal IgG antibodyagainst human EGFR1 (mAb528) for 1 hour at room temperature to allowantibodies to bind to the minicells. After incubation, minicells werewashed three times each with 1 mL of PBS (pH 7.4) to remove any unboundantibody. Minicells (1e08) were then analyzed by Western blot using anHRP-conjugated rabbit anti-mouse polyclonal antibody as the secondaryantibody. The Western Blot is shown in FIG. 4. Specific binding of thesecondary antibody was visualized using an Amersham ECL Detection Kit(GE Healthcare). Mouse anti-EGFR antibody (100 ng) was loaded as apositive control (lane after 12B5).

As shown in FIG. 4, the Fc region of the HRP-conjugated rabbitanti-mouse secondary antibody was bound by the Protein A and Protein Gfusion proteins in the 13B7 and 13C4 minicells (49.1 kDa and 38.5 kDa,respectively), independent of the EGFR1 antibody. In addition, the Fabregion of the secondary antibody binds to and detects the intact EGFR1antibody (˜150 kDa) bound to the 13B7 and 13C4 minicells.

Example 4

Minicells binding and displaying the anti-human EGFR1 antibody mAb528are selectively targeted to EGFR1-expressing human non-small cell lungcarcinoma cell line H460 in vitro. Minicells expressing and displayingLpp-OmpA-Protein G (13C4) and the appropriate Lpp-OmpA-Protein Gdeficient minicell control (12B4) were stained with themembrane-specific fluorescent imaging agent FM-143 for 1 hour and thenwashed 3 times each in an equal volume of 1× PBS. Following staining,13C4 minicells (expressing the Lpp-OmpA-Protein G fusion) wereco-incubated with an excess of mAb528 or species/isotype matched controlantibody against Keyhole Limpet Hemocyanin (KLH; not present inmammalian cells) to allow binding of antibodies to the surface of 13C4minicells. As an additional control, 12B4 minicells, which do notexpress Lpp-OmpA-Protein G fusion, were also co-incubated with an equalconcentration of mAb528. Following antibody binding, samples were washedthree times each and then allowed to incubate with cultured H460 cellsat a minicell to H460 cell ratio of 5000:1 for 2 hours. Following the 2hour incubation, cells were washed three times each in cell culturemedium and then visualized for the uptake of fluorescent minicells usingfluorescence microscopy. The fluorescence microscopy results are shownin FIG. 5.

As shown in the left panel of FIG. 5, minicells that did not expressLpp-OmpA-Protein G (12B4) did not bind the EGFR1-targeting antibody andwere not efficiently internalized by EGFR1-expressing H460 cells. Middlepanel of FIG. 5 shows that minicells expressing Lpp-OmpA-Protein G(13C4) bound the EGFR1-targeting antibody and were readily internalizedby EGFR1-expressing H460 cells, demonstrating targeting-dependentuptake. Right panel of FIG. 5 shows that minicells expressingLpp-OmpA-Protein G (13C4) and displaying a non-specific isotype matchedcontrol antibody (antibody targets KLH; not expressed in H460 cells)were not efficiently internalized by EGFR1-expressing H460 cells,demonstrating a need for specific targeting.

Example 5

Lpp-OmpA-Protein A 2 Fc (Protein A) minicells with a fusion protein werepurified after growth of bacteria at 30° C. using a 1.0 micron cut-offfilter instead of low speed centrifugation to enrich for minicells, andthen by using a Ficoll density gradient instead of sucrose. The ProteinA portion of the fusion protein is capable of binding antibodies throughtheir Fc-regions but is defective in F(ab′)₂ binding and resistant toOmpT protease. Following purification, Protein A minicells (expressingLpp-OmpA-Protein A 2 Fc) were stained with the fluorescent imaging agentCFSE (carboxyfluorescein diacetate, succinimidyl ester) and thendecorated with anti-human EGF receptor, anti-KLH or anti-human CD123(IL-3 receptor; not expressed by H460) antibodies as described inExample 4. Following removal of excess unbound antibody, the minicellswere incubated with H460 cells (human non-small cell lung carcinoma cellline expressing EGFR1) for 40 minutes and washed. Tumor cellinternalization of fluorescent minicells was determined by fluorescencemicroscopy. FIG. 6 shows fluorescence microscope images of H460 cellmonolayers incubated with minicells decorated with various antibodieswith prominent EGFR1 targeted minicell uptake demonstrated in FIG. 6Aversus anti-KLH (FIG. 6B) or anti-CD123 (FIG. 6C) targeted minicells.The no antibody control also demonstrated no uptake as expected (FIG.6D). In the same experiment outlined for FIG. 6, the results of relativeminicell uptake measured quantitatively by FACS analysis of trypsinizedcells are shown in FIG. 7.

TABLES

TABLE 1 Cross-linking target(s) Cross-linking reagent(s) Purpose(s)Amine to amine disuccinimidyl glutarate (DSG), disuccinimidyl Used tocross-link (homobifunctional) suberate (DSS),bis(sulfosuccinimidyl)suberate (BS3), Fc-regions totris(succinimidyl)aminotriacetate (TSAT), BS(PEG)5, polypeptides,BS(PEG)9, Lomant's reagent (DSP), 3,3′- peptides, DARPins,dithiobis(sulfosuccinimidylpropionate) (DTSSP), and other amine-disuccinimidyl tartrate (DST), Bis[2- containing conjugates(succinimidooxycarbonyloxy)ethyl]sulfone in non-selective (BSOCOES),ethylene glycol amino acid positions. bis[succinimidylsuccinate] (EGS),ethylene glycol Also used to attach bis[sulfosuccinimidylsuccinate](Sulfo-EGS), dimethyl Fc regions to DNA adipimidate (DMA), dimethylpimelimidate (DMP), aptamers when the dimethyl suberimidate (DMS),dimethyl 3,3′- aptamers contain a dithiobispropionimidate (DTBP),1,5-difluoro-2,4- primary amine. dinitrobenzene (DFDNB) Sulfhydryl toMaleimides (BMOE, BMB, BMH, TMEA, Used to cross-link sulfhydrylBM[PEG]2, BM[PEG]3, BMBD, and DTME), Fc-regions to (homobifunctional)Pyridyldthiols (DPDPB), vinylsulfone polypeptides, peptides, DARPins,and other amine- containing conjugates in selective fashion throughnaturally occurring or recombinantly engineered cysteine residues withinboth the Fc region as well as the molecule to be conjugated. Also usedto attach cysteine containing Fc regions to DNA aptamers when theaptamers and Fc region both contain a sulfhydryl group. Non-selectiveBis-[b-(4-Azidosalicylamido)ethyl]disulfide (BASED) Used to cross-link(homobifunctional) Fc-regions to polypeptides, peptides, DARPins,amine-containing conjugates, carbohydrates, aptamers, nucleic acids, andhormones in non-selective fashion. Amine to sufhydrylN-(a-Maleimidoacetoxy) succinimide ester (AMAS), Used to cross-link(heterobifunctional) BMPS, GMBS, Sulfo-GMBS, MBS, Sulfo-MBS, Fc-regionsto Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1- polypeptides,carboxylate (SMCC), Sulfosuccinimidyl-4-(N- peptides, DARPins,maleimidomethyl)cyclohexane-1-carboxylate (Sulfo- and other amine-SMCC), EMCS, Sulfo-EMCS, SMPB, Sulfo-SMPB, containing conjugates SMPH,LC-SMCC, Sulfo-KMUS, SM(PEG)2, in selective fashion SM(PEG)4, SM(PEG)6,SM(PEG)8, SM(PEG)12, through naturally SM(PEG)24, SPDP, LC-SPDP,Sulfo-LC-SPDP, occurring or SMPT, Sulfo-SMPT, SIA, SBAP, SIAB,Sulfo-SIAB, recombinantly engineered cysteine residues wherein thecysteine is present in the Fc region or the conjugate molecule. Alsoused to attach amine containing Fc regions to DNA aptamers and othernucleic acids (e.g. siRNA) when the nucleic acids contain a sulfhydrylgroup. Conversely, listed reagents can be used to attach cysteinecontaining Fc regions to aptamers or other nucleic acid molecules (e.g.siRNA) that contain primary amines. Amine to non-N-Hydroxysuccinimidyl-4-azidosalicylic acid (NHS- Used to cross-linkselective ASA), ANB-NOS, Sulfo-HSAB, Sulfo-NHS-LC-ASA, Fc-regions toSANPAH, Sulfo-SANPAH, Sulfo-SFAD, Sulfo- polypeptides, SAND, Sulfo-SAED,succinimidyl-diazirine (SDA), peptides, DARPins, Sulfo-SDA, LC-SDA,Sulfo-LC-SDA and other amine- containing conjugates in semi-selectivefashion through naturally occurring or engineered amine groups. Alsoused to attach amine containing Fc regions to DNA aptamers and othernucleic acids (e.g., siRNA). Amine to carboxyl Carbodiimides(dicyclohexylcarbodiimide [DCC], 1- Used to cross-linkEthyl-3-[3-dimethylaminopropyl]carbodiimide Fc-regions to hydrochloride[EDC or EDAC]) polypeptides, peptides, DARPins, and other amine-containing conjugates in selective fashion through the carboxy terminusof either the Fc region or the conjugate. Sulfhydryl to non-Pyridyldithiol/Aryl Azide (ADPD) Used to cross-link selective Fc-regionsto polypeptides, peptides, DARPins, and other sulfhydryl- containingconjugates in semi-selective fashion through naturally occurring orengineered sulfhydryl groups. Also used to attach sulfhydryl containingFc regions to DNA aptamers and other nucleic acids (e.g., siRNA).Sulfhydryl to Maleimide/Hydrazide, BMPH, 3,3′-N-[e- Used to cross-linkcarbohydrate Maleimidocaproic acid] hydrazide, trifluoroaceticsulfhydryl containing acid salt (EMCH), MPBH, KMUH Fc-regions tocarbohydrates. Hydroxyl to Isocyanate/Malemide (PMPI) Used to cross-linksulfydryl sulfhydryl containing Fc-regions to nucleic acids and otherconjugate molecules containing free hydroxyls. Amine to DNA NHSester/Psoralen (SPB) Used to cross-link Fc-regions to nucleic acids.

TABLE 2 Description of sequences provided in the sequence listing SEQ IDPlasmid NO. ORF 1 ORF 2 ORF 3 Name 1 Lpp-OmpA-Protein A — — pVX-119 2Lpp-OmpA-Protein G — — pVX-120 3 Lpp-OmpA-Protein G cLLO — pVX-127 4Lpp-OmpA-Protein G sLLO — pVX-175 5 Lpp-OmpA-Protein G sLLOpH — pVX-1766 Lpp-OmpA-Protein G PFO — pVX-177 7 Lpp-OmpA-ProteinG Diphtheria Toxin— pVX-199 Fragment A with native signal secretion signal sequence 8Lpp-OmpA-ProteinG Diphtheria Toxin — pVX-198 Fragment A with nosecretion signal sequence 9 Lpp-OmpA-ProteinG Gelonin — pVX-200 10Lpp-OmpA-Protein G cLLO Diphtheria Toxin pVX-195 Fragment A with nativesignal secretion sequence 11 Lpp-OmpA-Protein G sLLO Diphtheria ToxinpVX-180 Fragment A with native signal secretion sequence 12Lpp-OmpA-Protein G sLLOpH Diphtheria Toxin pVX-192 Fragment A withnative signal secretion sequence 13 Lpp-OmpA-Protein G PFO DiphtheriaToxin pVX-184 Fragment A with native signal secretion sequence 14Lpp-OmpA-Protein G cLLO Diphtheria Toxin pVX-158 Fragment A with nosignal sequence 15 Lpp-OmpA-Protein G sLLO Diphtheria Toxin pVX-179Fragment A with no signal sequence 16 Lpp-OmpA-Protein G sLLOpHDiphtheria Toxin pVX-191 Fragment A with no signal sequence 17Lpp-OmpA-Protein G PFO Diphtheria Toxin pVX-183 Fragment A with nosignal sequence 18 Lpp-OmpA-Protein G cLLO Gelonin pVX-196 19Lpp-OmpA-Protein G sLLO Gelonin pVX-181 20 Lpp-OmpA-Protein G sLLOpHGelonin pVX-193 21 Lpp-OmpA-Protein G PFO Gelonin pVX-185 33 PFOsL462F(PFOf) NA NA NA 35 PFOsG137Q (PFOq) NA NA NA 37 PFOsH438Y (PFOy) NA NANA 39 Lpp-OmpA-Protein A- NA NA NA 2Fc 41 Lpp-OmpA-Protein A NA NA NA2Fc-OTR (OmpT resistant) SEQ ID NO. Protein Name 22 Lpp-OmpA-ProteinG 23Lpp-OmpA-ProteinA 24 Gelonin 25 Diphtheria toxin Fragment A with nativesignal sequence 26 Diphtheria toxin Fragment A lacking signal sequence27 Perfringolysin O (PFO; from S et al.) 28 Perfringolysin O (PFO; fromTweeten et al.) 28 Listeriolysin O (LLO) 29 Listeriolysin O lackingsignal sequence (cLLO) 30 Listeriolysin O (stabilized; sLLO) 31Listeriolysin O (pH stabilized; sLLOpH) 34 Perfringolysin O (from S etal., point mutant)—PFOsL462F (PFOf) 36 Perfringolysin O (from S et al.,point mutant)—PFOsG137Q (PFOq) 38 Perfringolysin O (from S et al., pointmutant)—PFOsH438Y (PFOy) 40 Lpp-OmpA-Protein A-2Fc—No F(ab) binding 42Lpp-OmpA-Protein A 2Fc-OTR (OmpT resistant)—No F(ab) binding

What is claimed is:
 1. A bacterial minicell, comprising: (i) anFc-binding fusion protein displayed on the surface of the minicell,wherein the Fc-binding fusion protein comprises a) an outer membraneanchoring domain and b) an Fc binding portion of Protein A, wherein theFc-binding portion of Protein A has no F(ab')₂ binding capability andcomprises no cleavage sites by OmpT protease; (ii) one or more bioactivemolecules; and (iii) one or more Fc-containing targeting molecules boundto said Fc binding portion, wherein said one or more Fc-containingtargeting molecules recognize a eukaryotic antigen.
 2. The minicell ofclaim 1, wherein at least one of the one or more bioactive molecules isa protein toxin.
 3. The minicell of claim 2, wherein said protein toxinis selected from the group consisting of gelonin, diphtheria toxinfragment A, diphtheria toxin fragment A/B, tetanus toxin, E. coli heatlabile toxin LTI, E. coli heat labile toxin LTII, cholera toxin, C.perfringes iota toxin, Pseudomonas exotoxin A, shiga toxin, anthraxtoxin, MTX (B. sphaericus mosquilicidal toxin), perfringolysin O,streptolysin, barley toxin, mellitin, anthrax toxins LF and EF,adenylate cyclase toxin, botulinolysin B, botulinolysin E3,botulinolysin C, botulinum toxin A, cholera toxin, clostridium toxins A,B, and alpha, ricin, shiga A toxin, shiga-like A toxin, cholera A toxin,pertussis S1 toxin, E. coli heat labile toxin (LTB), pH stable variantsof listeriolysin O, thermostable variants of listeriolysin O, pH andthermostable variants of listeriolysin O, streptolysin O, streptolysin Oc, streptolysin O e, sphaericolysin, anthrolysin O, cereolysin,thuringiensilysin O, weihenstephanensilysin, alveolysin, brevilysin,butyriculysin, tetanolysin O, novyilysin, lectinolysin, pneumolysin,mitilysin, pseudopneumolysin, suilysin, intermedilysin, ivanolysin,seeligeriolysin O, vaginolysin, pyolysin, and any combination thereof.4. The minicell of claim 1, wherein at least one of the one or morebioactive molecules is a therapeutic small molecule drug.
 5. Theminicell of claim 4, wherein said therapeutic small molecule drug isselected from the group consisting of DNA damaging agents, agents thatinhibit DNA synthesis, microtubule and tubulin binding agents,anti-metabolites, inducers of oxidative damage, anti-angiogenics,endocrine therapies, anti-estrogens, immuno-modulators, histonedeacetylase inhibitors, inhibitors of signal transduction, inhibitors ofheat shock proteins, retinoids, inhibitors of growth factor receptors,anti-mitotic compounds, anti-inflammatories, cell cycle regulators,transcription factor inhibitors, and apoptosis inducers, and anycombination thereof.
 6. The minicell of claim 1, wherein at least one ofthe one or more bioactive molecules is a therapeutic nucleic acid. 7.The minicell of claim 1, wherein at least one of the one or morebioactive molecules is a therapeutic polypeptide.
 8. The minicell ofclaim 1, wherein at least one of the one or more bioactive molecules isa combination of a small molecule drug and a therapeutic nucleic acid.9. The minicell of claim 1, wherein at least one of the one or moreFc-containing targeting molecules is specific for a tumor cell surfacemolecule.
 10. The minicell of claim 1, wherein at least one of the oneor more Fc-containing targeting molecules is specific for an endothelialcell surface molecule.
 11. The minicell of claim 1, wherein at least oneof the one or more Fc-containing targeting molecules is specific for atarget common to both a tumor cell and an endothelial cell.
 12. Theminicell of claim 1, wherein said minicell further comprises anendosomal escape agent.
 13. A composition, comprising the minicell ofclaim 1 and a pharmaceutically acceptable carrier.
 14. The minicell ofclaim 1, wherein at least one of the one or more bioactive molecules isa protein from an infectious agent.
 15. The minicell of claim 14,wherein at least one of the one or more Fc-containing targetingmolecules is specific for a professional antigen presenting cell. 16.The minicell of claim 15, wherein said professional antigen presentingcell is a eukaryotic dendritic cell or macrophage.
 17. The minicell ofclaim 1, wherein at least one of the one or more bioactive molecules isa protein antigen from a tumor.
 18. The minicell of claim 17, wherein atleast one of the one or more Fc-containing targeting molecules isspecific for a eukaryotic dendritic cell, eosinophil, neutrophil,basophil, T-cell, B-cell, mast cell, or macrophage.
 19. The minicell ofclaim 14, wherein said minicell further comprises an endosomal escapeagent, or an immunomodulatory adjuvant.
 20. A composition, comprisingthe minicell of claim 14 and a pharmaceutically acceptable carrier. 21.The minicell of claim 1, wherein the Fc-binding fusion protein furthercomprises an autotransporter of type 5a secretion system.
 22. Theminicell of claim 1, wherein the minicell is fully intact.
 23. Theminicell of claim 1, wherein the Fc-binding portion of Protein Acomprises an amino acid substitution of glycine to alanine at glycine29.
 24. The minicell of claim 5, wherein the immune-modulators areToll-like receptor agonists or antagonists.